Developmental regulation of MURF ubiquitin ligases and autophagy proteins nbr1, p62/SQSTM1 and LC3 during cardiac myofibril assembly and turnover.

PubMed

Perera, Sue; Holt, Mark R; Mankoo, Baljinder S; Gautel, Mathias

2011-03-01

The striated muscle-specific tripartite motif (TRIM) proteins TRIM63/MURF1, TRIM55/MURF2 and TRIM54/MURF3 can function as ubiquitin E3 ligases in ubiquitin-mediated muscle protein turnover. Despite their well-characterised roles in muscle atrophy, the dynamics of MURF expression in the development and early postnatal adaptation of striated muscle is largely unknown. Here, we show that MURF2 is expressed at the very onset of mouse cardiac differentiation at embryonic day 8.5, and represents a sensitive marker for differentiating myocardium. During cardiac development, expression shifts from the 50 kDa to the 60 kDa A-isoform, which dominates postnatally. In contrast, MURF1 shows strong postnatal upregulation and MURF3 is not significantly expressed before birth. MURF2 expression parallels that of the autophagy-associated proteins LC3, p62/SQSTM1 and nbr1. SiRNA knockdown of MURF2 in neonatal rat cardiomyocytes disrupts posttranslational microtubule modification and myofibril assembly, and is only partly compensated by upregulation of MURF3 but not MURF1. Knockdown of both MURF2 and MURF3 severely disrupts the formation of ordered Z- and M-bands, likely by perturbed tubulin dynamics. These results suggest that ubiquitin-mediated protein turnover and MURF2 in particular play an unrecognised role in the earliest steps of heart muscle differentiation, and that partial complementation of MURF2 deficiency is afforded by MURF3. Copyright © 2010 Elsevier Inc. All rights reserved.

Nuclear 82-kDa choline acetyltransferase decreases amyloidogenic APP metabolism in neurons from APP/PS1 transgenic mice.

PubMed

Albers, Shawn; Inthathirath, Fatima; Gill, Sandeep K; Winick-Ng, Warren; Jaworski, Ewa; Wong, Daisy Y L; Gros, Robert; Rylett, R Jane

2014-09-01

Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to β-amyloid peptides (Aβ), cholinergic neuron loss with decreased choline acetyltransferase (ChAT) activity, and cognitive dysfunction. Both 69-kDa ChAT and 82-kDa ChAT are expressed in cholinergic neurons in human brain and spinal cord with 82-kDa ChAT localized predominantly to neuronal nuclei, suggesting potential alternative functional roles for the enzyme. By gene microarray analysis, we found that 82-kDa ChAT-expressing IMR32 neural cells have altered expression of genes involved in diverse cellular functions. Importantly, genes for several proteins that regulate APP processing along amyloidogenic and non-amyloidogenic pathways are differentially expressed in 82-kDa ChAT-containing cells. The predicted net effect based on observed changes in expression patterns of these genes would be decreased amyloidogenic APP processing with decreased Aβ production. This functional outcome was verified experimentally as a significant decrease in BACE1 protein levels and activity and a concomitant reduction in the release of endogenous Aβ1-42 from neurons cultured from brains of AD-model APP/PS1 transgenic mice. The expression of 82-kDa ChAT in neurons increased levels of GGA3, which is involved in trafficking BACE1 to lysosomes for degradation. shRNA-induced decreases in GGA3 protein levels attenuated the 82-kDa ChAT-mediated decreases in BACE1 protein and activity and Aβ1-42 release. Evidence that 82-kDa ChAT can enhance GGA3 gene expression is shown by enhanced GGA3 gene promoter activity in SN56 neural cells expressing this ChAT protein. These studies indicate a novel relationship between cholinergic neurons and APP processing, with 82-kDa ChAT acting as a negative regulator of Aβ production. This decreased formation of Aβ could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to

Anti-LC1 autoantibodies in patients with chronic hepatitis C virus infection.

PubMed

Béland, Kathie; Lapierre, Pascal; Marceau, Gabriel; Alvarez, Fernando

2004-03-01

Various autoantibodies have been reported in patients chronically infected by hepatitis C virus. 2% to 10% of theses patients have anti-liver-kidney microsome type 1 (anti-LKM1) autoantibodies. In type 2 autoimmune hepatitis, anti-LKM1 autoantibodies are frequently associated with anti-liver-cytosol type 1 (anti-LC1) autoantibodies. To determine the prevalence of anti-LC1 autoantibodies in a hepatitis C-positive population and characterize their reactivity. 146 patients suffering from liver diseases, of which 99 were chronically infected by hepatitis C virus, were tested by Western blotting and immunoprecipitation to detect and characterize anti-LC1 autoantibodies. 12% of this hepatitis C population had anti-LC1 autoantibodies. LC1 positivity by Western blotting was 30% of LC1+ sera. Epitopes were found throughout the protein but linear epitopes were situated in the 395-541 amino acid region of formiminotransferase cyclodeaminase. Three putative conformational epitopes were identified by phage display. Anti-LC1 autoantibodies are as prevalent as anti-LKM1 autoantibodies in patients infected with hepatitis C virus and their production is not dependent of anti-LKM1 autoantibodies formation. Autoantibody reactivity against the anti-LC1 antigen is different in hepatitis C than in type 2 autoimmune hepatitis. Anti-LC1 autoantibodies can now be regarded as a serological marker of autoimmunity in chronic hepatitis C infection.

During cooled storage the extender influences processed autophagy marker light chain 3 (LC3B) of stallion spermatozoa.

PubMed

Bolaños, J M Gallardo; Morán, A Miró; da Silva, C M Balao; Dávila, M Plaza; Muñoz, P Martín; Aparicio, I M; Tapia, J A; Ferrusola, C Ortega; Peña, F J

2014-02-01

To investigate the role of the processed autophagy marker light chain 3 (LC3B) protein in sperm survival in stallion semen processing during cooled storage, split ejaculates were diluted in two different extenders, KMT and INRA 96, and LC3B processing and sperm quality evaluated during incubation at 5°C for five days. After 3 days of incubation there was a drop in total motility in both extenders, although the percentage of progressive motile sperm was greater (P<0.05) in samples extended in INRA96. On Day 5 of cooled storage all sperm parameters decreased significantly independent of the extender, however, samples extended in INRA 96 maintained motility values while those extended in KMT had a further decrease in motility compared with data collected on Day 3 of incubation. The percentage of live sperm decreased over the time of incubation, but only in samples incubated in KMT. The extender had a marked effect in LC3B processing during cooled storage. Spermatozoa maintained in KMT extender did not exhibit LC3B processing, while in spermatozoa incubated in INRA96 there was an increase (P<0.01) in LC3B processing after 5 days of cooled storage. Stallion spermatozoa experience LC3B turnover during cooled storage, however, the extent depends on the extender used. Apparently LC3B turnover is associated with enhanced survival. Copyright © 2014 Elsevier B.V. All rights reserved.

Clinicopathological Correlations of Autophagy-related Proteins LC3, Beclin 1 and p62 in Gastric Cancer.

PubMed

Masuda, G O; Yashiro, Masakazu; Kitayama, Kishu; Miki, Yuichiro; Kasashima, Hiroaki; Kinoshita, Haruhito; Morisaki, Tamami; Fukuoka, Tatshunari; Hasegawa, Tsuyoshi; Sakurai, Katsunobu; Toyokawa, Takahiro; Kubo, Naoshi; Tanaka, Hiroaki; Muguruma, Kazuya; Masaichi, Ohira; Hirakawa, Kosei

2016-01-01

This study evaluated the clinicopathological significance of autophagy, an intracellular degradation system, in gastric cancer. The expression levels of three autophagy-related proteins, namely light chain 3 (LC3), Beclin 1 and p62, were analyzed by immunohistochemistry using samples from 510 patients with primary gastric cancer. LC3, Beclin 1, and p62 expression was positive in 79 (15.5%), 126 (24.7%) and 251 (49.2%) out of 510 carcinomas, respectively. Autophagy was defined when samples were positive for at least two out of the three proteins. Autophagy-positive cases were 113 (22.1%) out of the 510. Autophagy determined by LC3, Beclin 1, and p62 significantly correlated with lymph node metastasis, vessel invasion, and hepatic metastasis. A Kaplan-Meier survival curve showed that autophagy was significantly associated with poor survival of patients with gastric cancer, especially for those with disease at stage I. Multivariate analysis indicated that autophagy was an independent prognostic factor. Autophagy promotes the progression of gastric cancer at an early clinical stage. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Three Members of the LC8/DYNLL Family Are Required for Outer Arm Dynein Motor Function

PubMed Central

Tanner, Christopher A.; Rompolas, Panteleimon; Patel-King, Ramila S.; Gorbatyuk, Oksana; Wakabayashi, Ken-ichi; Pazour, Gregory J.

2008-01-01

The highly conserved LC8/DYNLL family proteins were originally identified in axonemal dyneins and subsequently found to function in multiple enzyme systems. Genomic analysis uncovered a third member (LC10) of this protein class in Chlamydomonas. The LC10 protein is extracted from flagellar axonemes with 0.6 M NaCl and cofractionates with the outer dynein arm in sucrose density gradients. Furthermore, LC10 is specifically missing only from axonemes of those strains that fail to assemble outer dynein arms. Previously, the oda12-1 insertional allele was shown to lack the Tctex2-related dynein light chain LC2. The LC10 gene is located ∼2 kb from that of LC2 and is also completely missing from this mutant but not from oda12-2, which lacks only the 3′ end of the LC2 gene. Although oda12-1 cells assemble outer arms that lack only LC2 and LC10, this strain exhibits a flagellar beat frequency that is consistently less than that observed for strains that fail to assemble the entire outer arm and docking complex (e.g., oda1). These results support a key regulatory role for the intermediate chain/light chain complex that is an integral and highly conserved feature of all oligomeric dynein motors. PMID:18579685

Prognostic value of the autophagy markers LC3 and p62/SQSTM1 in early-stage non-small cell lung cancer.

PubMed

Schläfli, Anna M; Adams, Olivia; Galván, José A; Gugger, Mathias; Savic, Spasenija; Bubendorf, Lukas; Schmid, Ralph A; Becker, Karl-Friedrich; Tschan, Mario P; Langer, Rupert; Berezowska, Sabina

2016-06-28

Autophagy is a cellular degrading process that promotes tumor cell survival or cell death in cancer, depending on the progress of oncogenesis. Protein light chain 3 (LC3) and p62/SQSTM1 (p62) are associated with autophagosomal membranes that engulf cytoplasmic content for subsequent degradation. We studied LC3 and p62 expression using immunohistochemistry in a large cohort of 466 stage I/II non-small cell lung cancer (NSCLC) using a tissue microarray. We evaluated dot-like cytoplasmic expression of LC3 and dot-like, cytoplasmic and nuclear staining for p62 in relation to clinico-pathological parameters.LC3 expression correlated with all p62 patterns, as those correlated among each other (p < 0.001 each). There was no correlation with stage, age or gender. A combination of high LC3/high p62 dot-like staining (suggesting impaired autophagy) showed a trend for better outcome (p = 0.11). Interestingly, a combined low cytoplasmic/low nuclear p62 expression regardless of dot-like staining was an independent prognostic factor for longer survival (p = 0.006; HR=1.96), in addition to tumor stage (p = 0.004; HR=1.4).The autophagy markers LC3 and p62 are differentially expressed in NSCLC, pointing towards a biologically significant role. High LC3 levels seem to be linked to lower tumor aggressiveness, while high general p62 expression was significantly associated with aggressive tumor behavior.

Prognostic value of the autophagy markers LC3 and p62/SQSTM1 in early-stage non-small cell lung cancer

PubMed Central

Schläfli, Anna M.; Adams, Olivia; Galván, José A.; Gugger, Mathias; Savic, Spasenija; Bubendorf, Lukas; Schmid, Ralph A.; Becker, Karl-Friedrich; Tschan, Mario P.; Langer, Rupert; Berezowska, Sabina

2016-01-01

Autophagy is a cellular degrading process that promotes tumor cell survival or cell death in cancer, depending on the progress of oncogenesis. Protein light chain 3 (LC3) and p62/SQSTM1 (p62) are associated with autophagosomal membranes that engulf cytoplasmic content for subsequent degradation. We studied LC3 and p62 expression using immunohistochemistry in a large cohort of 466 stage I/II non-small cell lung cancer (NSCLC) using a tissue microarray. We evaluated dot-like cytoplasmic expression of LC3 and dot-like, cytoplasmic and nuclear staining for p62 in relation to clinico-pathological parameters. LC3 expression correlated with all p62 patterns, as those correlated among each other (p < 0.001 each). There was no correlation with stage, age or gender. A combination of high LC3/high p62 dot-like staining (suggesting impaired autophagy) showed a trend for better outcome (p = 0.11). Interestingly, a combined low cytoplasmic/low nuclear p62 expression regardless of dot-like staining was an independent prognostic factor for longer survival (p = 0.006; HR=1.96), in addition to tumor stage (p = 0.004; HR=1.4). The autophagy markers LC3 and p62 are differentially expressed in NSCLC, pointing towards a biologically significant role. High LC3 levels seem to be linked to lower tumor aggressiveness, while high general p62 expression was significantly associated with aggressive tumor behavior. PMID:27250032

CGK733-induced LC3 II formation is positively associated with the expression of cyclin-dependent kinase inhibitor p21Waf1/Cip1 through modulation of the AMPK and PERK/CHOP signaling pathways.

PubMed

Wang, Yufeng; Kuramitsu, Yasuhiro; Baron, Byron; Kitagawa, Takao; Tokuda, Kazuhiro; Akada, Junko; Nakamura, Kazuyuki

2015-11-24

Microtubule-associated protein 1A/1B-light chain 3 (LC3)-II is essential for autophagosome formation and is widely used to monitor autophagic activity. We show that CGK733 induces LC3 II and LC3-puncta accumulation, which are not involved in the activation of autophagy. The treatment of CGK733 did not alter the autophagic flux and was unrelated to p62 degradation. Treatment with CGK733 activated the AMP-activated protein kinase (AMPK) and protein kinase RNA-like endoplasmic reticulum kinase/CCAAT-enhancer-binding protein homologous protein (PERK/CHOP) pathways and elevated the expression of p21Waf1/Cip1. Inhibition of both AMPK and PERK/CHOP pathways by siRNA or chemical inhibitor could block CGK733-induced p21Waf1/Cip1 expression as well as caspase-3 cleavage. Knockdown of LC3 B (but not LC3 A) abolished CGK733-triggered LC3 II accumulation and consequently diminished AMPK and PERK/CHOP activity as well as p21Waf1/Cip1 expression. Our results demonstrate that CGK733-triggered LC3 II formation is an initial event upstream of the AMPK and PERK/CHOP pathways, both of which control p21Waf1/Cip1 expression.

A high throughput liquid crystal light shutter for unpolarized light using polymer polarization gratings

NASA Astrophysics Data System (ADS)

Komanduri, Ravi K.; Lawler, Kris F.; Escuti, Michael J.

2011-05-01

We report on a broadband, diffractive, light shutter with the ability to modulate unpolarized light. This polarizer-free approach employs a conventional liquid crystal (LC) switch, combined with broadband Polarization Gratings (PGs) formed with polymer LC materials. The thin-film PGs act as diffractive polarizing beam-splitters, while the LC switch operates on both orthogonal polarization states simultaneously. As an initial experimental proof-of- concept for unpolarized light with +/-7° aperture, we utilize a commercial twisted-nematic LC switch and our own polymer PGs to achieve a peak transmittance of 80% and peak contrast ratio of 230:1. We characterize the optoelectronic performance, discuss the limitations, and evaluate its use in potential nonmechanical shutter applications (imaging and non-imaging).

Prognostic impact of Beclin 1, p62/sequestosome 1 and LC3 protein expression in colon carcinomas from patients receiving 5-fluorouracil as adjuvant chemotherapy.

PubMed

Park, Jae Myung; Huang, Shengbing; Wu, Tsung-Teh; Foster, Nathan R; Sinicrope, Frank A

2013-02-01

Autophagy is a cellular degradation process that can be activated in tumor cells to confer stress tolerance. During autophagy initiation and autophagosome formation, Beclin 1 binds microtubule-associated protein-1 light chain 3 (LC3I) that is converted to its membrane-bound form (LC3II) and interacts with the ubiquitin-binding protein p62/sequestosome 1 (SQSTM1). We determined the association of Beclin 1, LC3 and p62 protein expression with clinical outcome in resected stage II and III colon carcinomas (n = 178) from participants in 5-fluororuacil (5-FU)-based adjuvant therapy trials. The immunopercentage for each marker was determined and dichotomized for analysis with overall survival (OS) using Cox models. We found that autophagy markers localized to the tumor cell cytoplasm and showed increased expression relative to normal epithelial cells. Overexpression of Beclin 1, LC3 and p62 proteins were detected in 69%, 79% and 85% of tumors, respectively. Expression levels were not significantly associated with clinicopathological variables. In a multivariable analysis adjusting for tumor grade, stage and patient age, Beclin 1 overexpression was independently associated with worse OS [hazard ratio (HR), 1.82; 95% confidence interval (CI), 1.0-3.3; p = 0.042] in patients who received 5-FU-based adjuvant therapy. Neither LC3 nor p62 overexpression was prognostic. In conclusion, Beclin 1 overexpression was associated with reduced survival in colon cancer patients treated with adjuvant 5-FU. These data are consistent with preclinical evidence indicating that autophagy can protect colon cancer cells from 5-FU and support the targeting of autophagy for therapeutic advantage in this malignancy.

GeLC-MRM quantitation of mutant KRAS oncoprotein in complex biological samples.

PubMed

Halvey, Patrick J; Ferrone, Cristina R; Liebler, Daniel C

2012-07-06

Tumor-derived mutant KRAS (v-Ki-ras-2 Kirsten rat sarcoma viral oncogene) oncoprotein is a critical driver of cancer phenotypes and a potential biomarker for many epithelial cancers. Targeted mass spectrometry analysis by multiple reaction monitoring (MRM) enables selective detection and quantitation of wild-type and mutant KRAS proteins in complex biological samples. A recently described immunoprecipitation approach (Proc. Nat. Acad. Sci.2011, 108, 2444-2449) can be used to enrich KRAS for MRM analysis, but requires large protein inputs (2-4 mg). Here, we describe sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based enrichment of KRAS in a low molecular weight (20-25 kDa) protein fraction prior to MRM analysis (GeLC-MRM). This approach reduces background proteome complexity, thus, allowing mutant KRAS to be reliably quantified in low protein inputs (5-50 μg). GeLC-MRM detected KRAS mutant variants (G12D, G13D, G12V, G12S) in a panel of cancer cell lines. GeLC-MRM analysis of wild-type and mutant was linear with respect to protein input and showed low variability across process replicates (CV = 14%). Concomitant analysis of a peptide from the highly similar HRAS and NRAS proteins enabled correction of KRAS-targeted measurements for contributions from these other proteins. KRAS peptides were also quantified in fluid from benign pancreatic cysts and pancreatic cancers at concentrations from 0.08 to 1.1 fmol/μg protein. GeLC-MRM provides a robust, sensitive approach to quantitation of mutant proteins in complex biological samples.

Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1.

PubMed

Hirano, Satoshi; Uemura, Takefumi; Annoh, Hiromichi; Fujita, Naonobu; Waguri, Satoshi; Itoh, Takashi; Fukuda, Mitsunori

2016-01-01

MAP1LC3/LC3 (a mammalian ortholog family of yeast Atg8) is a ubiquitin-like protein that is essential for autophagosome formation. LC3 is conjugated to phosphatidylethanolamine on phagophores and ends up distributed both inside and outside the autophagosome membrane. One of the well-known functions of LC3 is as a binding partner for receptor proteins, which target polyubiquitinated organelles and proteins to the phagophore through direct interaction with LC3 in selective autophagy, and their LC3-binding ability is essential for degradation of the polyubiquitinated substances. Although a number of LC3-binding proteins have been identified, it is unknown whether they are substrates of autophagy or how their interaction with LC3 is regulated. We previously showed that one LC3-binding protein, TBC1D25/OATL1, plays an inhibitory role in the maturation step of autophagosomes and that this function depends on its binding to LC3. Interestingly, TBC1D25 seems not to be a substrate of autophagy, despite being present on the phagophore. In this study we investigated the molecular basis for the escape of TBC1D25 from autophagic degradation by performing a chimeric analysis between TBC1D25 and SQSTM1/p62 (sequestosome 1), and the results showed that mutant TBC1D25 with an intact LC3-binding site can become an autophagic substrate when TBC1D25 is forcibly oligomerized. In addition, an ultrastructural analysis showed that TBC1D25 is mainly localized outside autophagosomes, whereas an oligomerized TBC1D25 mutant rather uniformly resides both inside and outside the autophagosomes. Our findings indicate that oligomerization is a key factor in the degradation of LC3-binding proteins and suggest that lack of oligomerization ability of TBC1D25 results in its asymmetric localization at the outer autophagosome membrane.

V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation.

PubMed

Florey, Oliver; Gammoh, Noor; Kim, Sung Eun; Jiang, Xuejun; Overholtzer, Michael

2015-01-01

Recently a noncanonical activity of autophagy proteins has been discovered that targets lipidation of microtubule-associated protein 1 light chain 3 (LC3) onto macroendocytic vacuoles, including macropinosomes, phagosomes, and entotic vacuoles. While this pathway is distinct from canonical autophagy, the mechanism of how these nonautophagic membranes are targeted for LC3 lipidation remains unclear. Here we present evidence that this pathway requires activity of the vacuolar-type H(+)-ATPase (V-ATPase) and is induced by osmotic imbalances within endolysosomal compartments. LC3 lipidation by this mechanism is induced by treatment of cells with the lysosomotropic agent chloroquine, and through exposure to the Heliobacter pylori pore-forming toxin VacA. These data add novel mechanistic insights into the regulation of noncanonical LC3 lipidation and its associated processes, including LC3-associated phagocytosis (LAP), and demonstrate that the widely and therapeutically used drug chloroquine, which is conventionally used to inhibit autophagy flux, is an inducer of LC3 lipidation.

Regulation of the autophagy protein LC3 by phosphorylation

PubMed Central

Cherra, Salvatore J.; Kulich, Scott M.; Uechi, Guy; Balasubramani, Manimalha; Mountzouris, John; Day, Billy W.

2010-01-01

Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP+) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity. PMID:20713600

Effect of 14-kDa and 47-kDa protein molecules of age garlic extract on peritoneal macrophages.

PubMed

Daneshmandi, Saeed; Hajimoradi, Monire; Ahmadabad, Hasan Namdar; Hassan, Zuhair Mohammad; Roudbary, Maryam; Ghazanfari, Tooba

2011-03-01

Garlic (Allium sativum), traditionally being used as a spice worldwide, has different applications and is claimed to possess beneficial effects in several health ailments such as tumor and atherosclerosis. Garlic is also an immunomodulator and its different components are responsible for different properties. The present work aimed to assess the effect of protein fractions of garlic on peritoneal macrophages. 14-kDa and 47-kDa protein fractions of garlic were purified. Mice peritoneal macrophages were lavaged and cultured in a microtiter plate and exposed to different concentrations of garlic proteins. MTT assay was performed to evaluate the viability of macrophage. The amount of nitric oxide (NO) was detected in culture supernatants of macrophages by Griess reagent and furthermore, the cytotoxicity study of culture supernatants was carried out on WEHI-164 fibrosarcoma cell line as tumor necrosis factor-α bioassay. MTT assay results for both 14-kDa and 47-kDa protein fractions of stimulated macrophages were not significant (P > 0.05). Both 14-kDa and 47-kDa fractions significantly suppressed production of NO from macrophages (P = 0.007 and P = 0.003, respectively). Cytotoxicity of macrophages' supernatant on WEHI-164 fibrosarcoma cells was not affected by garlic protein fractions (P = 0.066 for 14-kDa and P = 0.085 for 47-kDa fractions). according to our finding, 14-kDa and 47-kDa fractions of aged garlic extract are able to suppress NO production from macrophages, which can be used as a biological advantage. These molecules had no cytotoxic effect on macrophages and do not increase tumoricidal property of macrophages.

RKIP phosphorylation–dependent ERK1 activation stimulates adipogenic lipid accumulation in 3T3-L1 preadipocytes overexpressing LC3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hahm, Jong Ryeal; Institute of Health Sciences, Gyeongsang National University School of Medicine, JinJu, 527-27; Ahmed, Mahmoud

3T3-L1 preadipocytes undergo adipogenesis in response to treatment with dexamethaxone, 1-methyl-3-isobutylxanthine, and insulin (DMI) through activation of several adipogenic transcription factors. Many autophagy-related proteins are also highly activated in the earlier stages of adipogenesis, and the LC3 conjugation system is required for formation of lipid droplets. Here, we investigated the effect of overexpression of green fluorescent protein (GFP)-LC3 fusion protein on adipogenesis. Overexpression of GFP-LC3 in 3T3-L1 preadipocytes using poly-L-lysine-assisted adenoviral GFP-LC3 transduction was sufficient to produce intracellular lipid droplets. Indeed, GFP-LC3 overexpression stimulated expression of some adipogenic transcription factors (e.g., C/EBPα or β, PPARγ, SREBP2). In particular, SREBP2 wasmore » highly activated in preadipocytes transfected with adenoviral GFP-LC3. Also, phosphorylation of Raf kinase inhibitory protein (RKIP) at serine 153, consequently stimulating extracellular-signal regulated kinase (ERK)1 activity, was significantly increased during adipogenesis induced by either poly-L-lysine-assisted adenoviral GFP-LC3 transduction or culture in the presence of dexamethasone, 1-methyl-3-isobutylxanthine, and insulin. Furthermore, RKIP knockdown promoted ERK1 and PPARγ activation, and significantly increased the intracellular accumulation of triacylglycerides in DMI-induced adipogenesis. In conclusion, GFP-LC3 overexpression in 3T3-L1 preadipocytes stimulates adipocyte differentiation via direct modulation of RKIP-dependent ERK1 activity. - Highlights: • Overexpression of GFP-LC3 in 3T3-L1 cells produces intracellular lipid droplets. • SREBP2 is highly activated in preadipocytes transfected with adenoviral GFP-LC3. • RKIP phosphorylation at serine 153 is significantly increased during adipogenesis. • RKIP knockdown promotes ERK1 and PPARγ activation during adipogenesis. • RKIP-dependent ERK1 activation increases

Productive Recognition of Factor IX by Factor XIa Exosites Requires Disulfide Linkage between Heavy and Light Chains of Factor XIa*

PubMed Central

Marcinkiewicz, Mariola M.; Sinha, Dipali; Walsh, Peter N.

2012-01-01

In the intrinsic pathway of blood coagulation factor XIa (FXIa) activates factor IX (FIX) by cleaving the zymogen at Arg145-Ala146 and Arg180-Val181 bonds releasing an 11-kDa activation peptide. FXIa and its isolated light chain (FXIa-LC) cleave S-2366 at comparable rates, but FXIa-LC is a very poor activator of FIX, possibly because FIX undergoes allosteric modification on binding to an exosite on the heavy chain of FXIa (FXIa-HC) required for optimal cleavage rates of the two scissile bonds of FIX. However preincubation of FIX with a saturating concentration of isolated FXIa-HC did not result in any potentiation in the rate of FIX cleavage by FXIa-LC. Furthermore, if FIX binding via the heavy chain exosite of FXIa determines the affinity of the enzyme-substrate interaction, then the isolated FXIa-HC should inhibit the rate of FIX activation by depleting the substrate. However, whereas FXIa/S557A inhibited FIX activation of by FXIa, FXIa-HC did not. Therefore, we examined FIX binding to FXIa/S557A, FXIa-HC, FXIa-LC, FXIa/C362S/C482S, and FXIa/S557A/C362S/C482S. The heavy and light chains are disulfide-linked in FXIa/S557A but not in FXIa/C362S/C482S and FXIa/S557A/C362S/C482S. In an ELISA assay only FXI/S557A ligated FIX with high affinity. Partial reduction of FXIa/S557A to produce heavy and light chains resulted in decreased FIX binding, and this function was regained upon reformation of the disulfide linkage between the heavy and the light chains. We therefore conclude that substrate recognition by the FXIa exosite(s) requires disulfide-linked heavy and light chains. PMID:22207756

The transcription factor Lc-Maf participates in Col27a1 regulation during chondrocyte maturation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mayo, Jaime L.; Holden, Devin N.; Barrow, Jeffery R.

2009-08-01

The transcription factor Lc-Maf, which is a splice variant of c-Maf, is expressed in cartilage undergoing endochondral ossification and participates in the regulation of type II collagen through a cartilage-specific Col2a1 enhancer element. Type XXVII and type XI collagens are also expressed in cartilage during endochondral ossification, and so enhancer/reporter assays were used to determine whether Lc-Maf could regulate cartilage-specific enhancers from the Col27a1 and Col11a2 genes. The Col27a1 enhancer was upregulated over 4-fold by Lc-Maf, while the Col11a2 enhancer was downregulated slightly. To confirm the results of these reporter assays, rat chondrosarcoma (RCS) cells were transiently transfected with anmore » Lc-Maf expression plasmid, and quantitative RT-PCR was performed to measure the expression of endogenous Col27a1 and Col11a2 genes. Endogenous Col27a1 was upregulated 6-fold by Lc-Maf overexpression, while endogenous Col11a2 was unchanged. Finally, in situ hybridization and immunohistochemistry were performed in the radius and ulna of embryonic day 17 mouse forelimbs undergoing endochondral ossification. Results demonstrated that Lc-Maf and Col27a1 mRNAs are coexpressed in proliferating and prehypertrophic regions, as would be predicted if Lc-Maf regulates Col27a1 expression. Type XXVII collagen protein was also most abundant in prehypertrophic and proliferating chondrocytes. Others have shown that mice that are null for Lc-Maf and c-Maf have expanded hypertrophic regions with reduced ossification and delayed vascularization. Separate studies have indicated that Col27a1 may serve as a scaffold for ossification and vascularization. The work presented here suggests that Lc-Maf may affect the process of endochondral ossification by participating in the regulation of Col27a1 expression.« less

NADH:ubiquinone oxidoreductase from bovine heart mitochondria. cDNA sequences of the import precursors of the nuclear-encoded 39 kDa and 42 kDa subunits.

PubMed Central

Fearnley, I M; Finel, M; Skehel, J M; Walker, J E

1991-01-01

The 39 kDa and 42 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria are nuclear-coded components of the hydrophobic protein fraction of the enzyme. Their amino acid sequences have been deduced from the sequences of overlapping cDNA clones. These clones were amplified from total bovine heart cDNA by means of the polymerase chain reaction, with the use of complex mixtures of oligonucleotide primers based upon fragments of protein sequence determined at the N-terminals of the proteins and at internal sites. The protein sequences of the 39 kDa and 42 kDa subunits are 345 and 320 amino acid residues long respectively, and their calculated molecular masses are 39,115 Da and 36,693 Da. Both proteins are predominantly hydrophilic, but each contains one or two hydrophobic segments that could possibly be folded into transmembrane alpha-helices. The bovine 39 kDa protein sequence is related to that of a 40 kDa subunit from complex I from Neurospora crassa mitochondria; otherwise, it is not related significantly to any known sequence, including redox proteins and two polypeptides involved in import of proteins into mitochondria, known as the mitochondrial processing peptidase and the processing-enhancing protein. Therefore the functions of the 39 kDa and 42 kDa subunits of complex I are unknown. The mitochondrial gene product, ND4, a hydrophobic component of complex I with an apparent molecular mass of about 39 kDa, has been identified in preparations of the enzyme. This subunit stains faintly with Coomassie Blue dye, and in many gel systems it is not resolved from the nuclearcoded 36 kDa subunit. Images Fig. 1. PMID:1832859

Light-directing chiral liquid crystal nanostructures: from 1D to 3D.

PubMed

Bisoyi, Hari Krishna; Li, Quan

2014-10-21

Endowing external, remote, and dynamic control to self-organized superstructures with desired functionalities is a principal driving force in the bottom-up nanofabrication of molecular devices. Light-driven chiral molecular switches or motors in liquid crystal (LC) media capable of self-organizing into optically tunable one-dimensional (1D) and three-dimensional (3D) superstructures represent such an elegant system. As a consequence, photoresponsive cholesteric LCs (CLCs), i.e., self-organized 1D helical superstructures, and LC blue phases (BPs), i.e., self-organized 3D periodic cubic lattices, are emerging as a new generation of multifunctional supramolecular 1D and 3D photonic materials in their own right because of their fundamental academic interest and technological significance. These smart stimuli-responsive materials can be facilely fabricated from achiral LC hosts by the addition of a small amount of a light-driven chiral molecular switch or motor. The photoresponsiveness of these materials is a result of both molecular interaction and geometry changes in the chiral molecular switch upon light irradiation. The doped photoresponsive CLCs undergo light-driven pitch modulation and/or helix inversion, which has many applications in color filters, polarizers, all-optical displays, optical lasers, sensors, energy-saving smart devices, and so on. Recently, we have conceptualized and rationally synthesized different light-driven chiral molecular switches that have very high helical twisting powers (HTPs) and exhibit large changes in HTP in different states, thereby enabling wide phototunability of the systems by the addition of very small amounts of the molecular switches into commercially available achiral LCs. The light-driven chiral molecular switches are based on well-recognized azobenzene, dithienylcyclopentene, and spirooxazine derivatives. We have demonstrated high-resolution and lightweight photoaddressable displays without patterned electronics on

LC3-mediated fibronectin mRNA translation induces fibrosarcoma growth by increasing connective tissue growth factor

PubMed Central

Ying, Lihua; Lau, Agatha; Alvira, Cristina M.; West, Robert; Cann, Gordon M.; Zhou, Bin; Kinnear, Caroline; Jan, Eric; Sarnow, Peter; Van de Rijn, Matt; Rabinovitch, Marlene

2009-01-01

Summary Previously, we related fibronectin (Fn1) mRNA translation to an interaction between an AU-rich element in the Fn1 3′ UTR and light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. Since human fibrosarcoma (HT1080) cells produce little fibronectin and LC3, we used these cells to investigate how LC3-mediated Fn1 mRNA translation might alter tumor growth. Transfection of HT1080 cells with LC3 enhanced fibronectin mRNA translation. Using polysome analysis and RNA-binding assays, we show that elevated levels of translation depend on an interaction between a triple arginine motif in LC3 and the AU-rich element in Fn1 mRNA. Wild-type but not mutant LC3 accelerated HT1080 cell growth in culture and when implanted in SCID mice. Comparison of WT LC3 with vector-transfected HT1080 cells revealed increased fibronectin-dependent proliferation, adhesion and invasion. Microarray analysis of genes differentially expressed in WT and vector-transfected control cells indicated enhanced expression of connective tissue growth factor (CTGF). Using siRNA, we show that enhanced expression of CTGF is fibronectin dependent and that LC3-mediated adhesion, invasion and proliferation are CTGF dependent. Expression profiling of soft tissue tumors revealed increased expression of both LC3 and CTGF in some locally invasive tumor types. PMID:19366727

Expanded test method for peptides >2 kDa employing immunoaffinity purification and LC-HRMS/MS.

PubMed

Thomas, Andreas; Walpurgis, Katja; Tretzel, Laura; Brinkkötter, Paul; Fichant, Eric; Delahaut, Philippe; Schänzer, Wilhelm; Thevis, Mario

2015-01-01

Bioactive peptides with an approximate molecular mass of 2-12 kDa are of considerable relevance in sports drug testing. Such peptides have been used to manipulate several potential performance-enhancing processes in the athlete's body and include for example growth hormone releasing hormones (sermorelin, CJC-1293, CJC-1295, tesamorelin), synthetic/animal insulins (lispro, aspart, glulisine, glargine, detemir, degludec, bovine and porcine insulin), synthetic ACTH (synacthen), synthetic IGF-I (longR(3) -IGF-I) and mechano growth factors (human MGF, modified human MGF, 'full-length' MGF). A combined initial test method using one analytical procedure is a desirable tool in doping controls and related disciplines as requests for higher sample throughput with utmost comprehensiveness preferably at reduced costs are constantly issued. An approach modified from an earlier assay proved fit-for-purpose employing pre-concentration of all target analytes by means of ultrafiltration, immunoaffinity purification with coated paramagnetic beads, nano-ultra high performance liquid chromatography (UHPLC) separation, and subsequent detection by means of high resolution tandem mass spectrometry. The method was shown to be applicable to blood and urine samples, which represent the most common doping control specimens. The method was validated considering the parameters specificity, recovery (11-69%), linearity, imprecision (<25%), limit of detection (5-100 pg in urine, 0.1-2 ng in plasma), and ion suppression. The analysis of administration study samples for insulin degludec, detemir, aspart, and synacthen provided the essential data for the proof-of-principle of the method. Copyright © 2015 John Wiley & Sons, Ltd.

mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines.

PubMed

Gómez-Sánchez, Rubén; Yakhine-Diop, Sokhna M S; Rodríguez-Arribas, Mario; Bravo-San Pedro, José M; Martínez-Chacón, Guadalupe; Uribe-Carretero, Elisabet; Pinheiro de Castro, Diana C J; Pizarro-Estrella, Elisa; Fuentes, José M; González-Polo, Rosa A

2016-06-01

We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes.

Method of preparing a tunable-focus liquid-crystal (LC) lens

NASA Astrophysics Data System (ADS)

Li, Xiaolong; Zhou, Zuowei; Ren, Hongwen

2018-02-01

A liquid crystal (LC) lens is prepared by controlling the alignment of a LC using a homogeneous polyimide (PI) layer and a homeotropic PI layer. The rubbed homogeneous PI layer has a concave surface and the homeotropic PI layer is flat. The LC sandwiched between the two PI layers obtains a hybrid alignment which has the largest gradient of refractive index (GRIN) distribution. The LC layer exhibits a lens character because of its convex shape. Since the effective refractive index of the LC is larger than that of the homogeneous PI, the LC lens can focus a light with the shortest focal length in the voltage-off state. By applying an external voltage, the LC molecules can be reoriented along the electric field. As a result, the focal length of the LC lens is reduced. The focal length of the LC lens can be tuned from 30 to 120 μm when the voltage is changed from 0 to 7 Vrms. This LC lens has the advantages of no threshold, low operating voltage, and simple fabrication.

All-optical liquid crystal spatial light modulators

NASA Astrophysics Data System (ADS)

Tabiryan, Nelson; Grozhik, Vladimir; Khoo, Iam Choon; Nersisyan, Sarik R.; Serak, Svetlana

2003-12-01

Nonlinear optical processes in liquid crystals (LC) can be used for construction of all-optical spatial light modulators (SLM) where the photosensitivity and phase modulating functions are integrated into a single layer of an LC-material. Such spatial light integrated modulators (SLIMs) cost only a fraction of the conventional LC-SLM and can be used with high power laser radiation due to high transparency of LC materials and absence of light absorbing electrodes on the substrates of the LC-cell constituting the SLIM. Recent development of LC materials the photosensitivity of which is comparable to that of semiconductors has led to using SLIM in schemes of optical anti-jamming, sensor protection, and image processing. All-optical processes add remarkable versatility to the operation of SLIM harnessing the wealth inherent to light-matter interaction phenomena.

Smooth muscle myosin isoform expression and LC20 phosphorylation in innate rat airway hyperresponsiveness.

PubMed

Gil, Fulvio R; Zitouni, Nedjma B; Azoulay, Eric; Maghni, Karim; Lauzon, Anne-Marie

2006-11-01

Four smooth muscle myosin heavy chain (SMMHC) isoforms are generated by alternative mRNA splicing of a single gene. Two of these isoforms differ by the presence [(+)insert] or absence [(-)insert] of a 7-amino acid insert in the motor domain. The rate of actin filament propulsion of the (+)insert SMMHC isoform, as measured in the in vitro motility assay, is twofold greater than that of the (-)insert isoform. We hypothesized that a greater expression of the (+)insert SMMHC isoform and greater regulatory light chain (LC(20)) phosphorylation contribute to airway hyperresponsiveness. We measured airway responsiveness to methacholine in Fischer hyperresponsive and Lewis normoresponsive rats and determined SMMHC isoform mRNA and protein expression, as well as essential light chain (LC(17)) isoforms, h-caldesmon, and alpha-actin protein expression in their tracheae. We also measured tracheal muscle strip contractility in response to methacholine and corresponding LC(20) phosphorylation. We found Fischer rats have more (+)insert mRNA (69.4 +/- 2.0%) (mean +/- SE) than Lewis rats (53.0 +/- 2.4%; P < 0.05) and a 44% greater content of (+)insert isoform relative to total myosin protein. No difference was found for LC(17) isoform, h-caldesmon, and alpha-actin expression. The contractility experiments revealed a greater isometric force for Fischer trachealis segments (4.2 +/- 0.8 mN) than Lewis (1.9 +/- 0.4 mN; P < 0.05) and greater LC(20) phosphorylation level in Fischer (55.1 +/- 6.4) than in Lewis (41.4 +/- 6.1; P < 0.05) rats. These results further support the contention that innate airway hyperresponsiveness is a multifactorial disorder in which increased expression of the fast (+)insert SMMHC isoform and greater activation of LC(20) lead to smooth muscle hypercontractility.

LcMCII-1 is involved in the ROS-dependent senescence of the rudimentary leaves of Litchi chinensis.

PubMed

Wang, Congcong; Lü, Peitao; Zhong, Silin; Chen, Houbin; Zhou, Biyan

2017-01-01

LcMCII - 1 is a type II metacaspase. Over-expression of LcMCII- 1 in Arabidopsis promoted ROS-dependent and natural senescence. Virus-induced LcMCII- 1 silencing delayed the ROS-dependent senescence of the rudimentary leaves of Litchi chinensis . Litchi is an evergreen woody fruit tree that is widely cultivated in subtropical and tropical regions. Its floral buds are mixed with axillary or apical panicle primordia, leaf primordia and rudimentary leaves. A low spring temperature is vital for litchi production as it promotes the abscission of the rudimentary leaves, which could otherwise prevent panicle development. Hence, climate change could present additional challenges for litchi production. We previously reported that reactive oxygen species (ROS) can substitute low-temperature treatment to induce the senescence of rudimentary leaves. We have now identified from RNA-Seq data a litchi type II metacaspase gene, LcMCII-1, that is responsive to ROS. Silencing LcMCII-1 by virus-induced gene silencing delayed ROS-dependent senescence. The ectopic over-expression of LcMCII-1 in transgenic Arabidopsis promoted ROS-dependent and natural senescence. Consistently, the transient expression of LcMCII-1 in tobacco leaf by agroinfiltration resulted in leaf yellowing. Our findings demonstrate that LcMCII-1 is positively involved in the regulation of rudimentary leaf senescence in litchi and provide a new target for the future molecular breeding of new cultivars that can set fruit in warmer climates.

White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein.

PubMed Central

Ballario, P; Vittorioso, P; Magrelli, A; Talora, C; Cabibbo, A; Macino, G

1996-01-01

The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction pathway. We report the cloning of the wc-1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle. The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain. We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation. Images PMID:8612589

Nanoliposomes protect against AL amyloid light chain protein-induced endothelial injury.

PubMed

Truran, Seth; Weissig, Volkmar; Ramirez-Alvarado, Marina; Franco, Daniel A; Burciu, Camelia; Georges, Joseph; Murarka, Shishir; Okoth, Winter A; Schwab, Sara; Hari, Parameswaran; Migrino, Raymond Q

2014-03-01

A newly-recognized pathogenic mechanism underlying light chain amyloidosis (AL) involves endothelial dysfunction and cell injury caused by misfolded light chain proteins (LC). Nanoliposomes (NL) are artificial phospholipid vesicles that could attach to misfolded proteins and reduce tissue injury. To test whether co-treatment with NL reduces LC-induced endothelial dysfunction and cell death. Abdominal subcutaneous adipose arterioles from 14 non-AL subjects were cannulated; dilator response to acetylcholine and papaverine were measured at baseline and following 1-hour exposure to LC (20 µg/mL, 2 purified from AL subjects' urine, 1 from human recombinant LC [AL-09]) ± NL (phosphatidylcholine/cholesterol/phosphatidic acid 70/25/5 molar ratio) or NL alone. Human aortic artery endothelial cells (HAEC) were exposed to Oregon Green-labeled LC ± NL for 24 hours and intracellular LC and apoptosis (Hoechst stain) were measured. Circular dichroism spectroscopy was performed on AL-09 LC ± NL to follow changes in secondary structure and protein thermal stability. LC caused impaired dilation to acetylcholine that was restored by NL (control - 94.0 ± 1.8%, LC - 65.0 ± 7.1%, LC + NL - 95.3 ± 1.8%, p ≤ 0.001 LC versus control or LC + NL). NL protection was inhibited by L-NG-nitroarginine methyl ester. NL increased the beta sheet structure of LC, reduced endothelial cell internalization of LC and protected against LC-induced endothelial cell death. LC induced human adipose arteriole endothelial dysfunction and endothelial cell death, which were reversed by co-treatment with NL. This protection may partly be due to enhancing LC protein structure and reducing LC internalization. Nanoliposomes represent a promising new class of agents to ameliorate tissue injury from protein misfolding diseases such as AL.

Possibility of the transformation of eEF-2 (100 kDa) to eEF-2 (65 kDa) in the peptide elongation process in vitro.

PubMed

Gajko, A; Sredzińska, K; Galasiński, W; Gindzieński, A

1999-02-16

Two active eEF-2 polypeptides of approximately 100 and 65 kDa were copurified from rat liver cells and separated. The fate of eEF-2 (100 kDa) during its binding to ribosomes and in the translocation step of the peptide elongation process was investigated. It was shown that eEF-2 (100 kDa) did not change its form during the process of binding to the ribosomes. In the postribosomal supernatant, obtained from the postincubation mixture of the elongation process, only eEF-2 (65 kDa) was found. These results suggest that the form of eEF-2 (100 kDa), when bound to the ribosome during the elongation process, is transformed to eEF-2 (65 kDa). Copyright 1999 Academic Press.

Photodynamic therapy with decacationic [60]fullerene monoadducts: effect of a light absorbing electron-donor antenna and micellar formulation.

PubMed

Yin, Rui; Wang, Min; Huang, Ying-Ying; Huang, Huang-Chiao; Avci, Pinar; Chiang, Long Y; Hamblin, Michael R

2014-05-01

We report the synthesis and anticancer photodynamic properties of two new decacationic fullerene (LC14) and red light-harvesting antenna-fullerene conjugated monoadduct (LC15) derivatives. The antenna of LC15 was attached covalently to C60>with distance of only <3.0 Ǻ to facilitate ultrafast intramolecular photoinduced-electron-transfer (for type-I photochemistry) and photon absorption at longer wavelengths. Because LC15 was hydrophobic we compared formulation in Cremophor EL micelles with direct dilution from dimethylacetamide. LC14 produced more (1)O2 than LC15, while LC15 produced much more HO·than LC14 as measured by specific fluorescent probes. When delivered by DMA, LC14 killed more HeLa cells than LC15 when excited by UVA light, while LC15 killed more cells when excited by white light consistent with the antenna effect. However LC15 was more effective than LC14 when delivered by micelles regardless of the excitation light. Micellar delivery produced earlier apoptosis and damage to the endoplasmic reticulum as well as to lysosomes and mitochondria. This team of authors report the synthesis and the photodynamic properties of two new derivatives for cancer treatment; one is a decacationic fullerene (LC14) and the other is a red light-harvesting antenna-fullerene conjugated monoadduct (LC15) utilizing a HeLa cell model. Copyright © 2014 Elsevier Inc. All rights reserved.

Monosialoganglioside-Containing Nanoliposomes Restore Endothelial Function Impaired by AL Amyloidosis Light Chain Proteins.

PubMed

Franco, Daniel A; Truran, Seth; Weissig, Volkmar; Guzman-Villanueva, Diana; Karamanova, Nina; Senapati, Subhadip; Burciu, Camelia; Ramirez-Alvarado, Marina; Blancas-Mejia, Luis M; Lindsay, Stuart; Hari, Parameswaran; Migrino, Raymond Q

2016-06-13

Light chain amyloidosis (AL) is associated with high mortality, especially in patients with advanced cardiovascular involvement. It is caused by toxicity of misfolded light chain proteins (LC) in vascular, cardiac, and other tissues. There is no treatment to reverse LC tissue toxicity. We tested the hypothesis that nanoliposomes composed of monosialoganglioside, phosphatidylcholine, and cholesterol (GM1 ganglioside-containing nanoliposomes [NLGM1]) can protect against LC-induced human microvascular dysfunction and assess mechanisms behind the protective effect. The dilator responses of ex vivo abdominal adipose arterioles from human participants without AL to acetylcholine and papaverine were measured before and after exposure to LC (20 μg/mL) with or without NLGM1 (1:10 ratio for LC:NLGM1 mass). Human umbilical vein endothelial cells were exposed for 18 to 20 hours to vehicle, LC with or without NLGM1, or NLGM1 and compared for oxidative and nitrative stress response and cellular viability. LC impaired arteriole dilator response to acetylcholine, which was restored by co-treatment with NLGM1. LC decreased endothelial cell nitric oxide production and cell viability while increasing superoxide and peroxynitrite; these adverse effects were reversed by NLGM1. NLGM1 increased endothelial cell protein expression of antioxidant enzymes heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 and increased nuclear factor, erythroid 2 like 2 (Nrf-2) protein. Nrf-2 gene knockdown reduced antioxidant stress response and reversed the protective effects of NLGM1. NLGM1 protects against LC-induced human microvascular endothelial dysfunction through increased nitric oxide bioavailability and reduced oxidative and nitrative stress mediated by Nrf-2-dependent antioxidant stress response. These findings point to a potential novel therapeutic approach for light chain amyloidosis. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

CRISPR/Cas9 Mediated GFP Knock-in at the MAP1LC3B Locus in 293FT Cells Is Better for Bona Fide Monitoring Cellular Autophagy.

PubMed

Wu, Zhiqiang; Zhao, Jinlin; Qiu, Minghan; Mi, Zeyun; Meng, Maobin; Guo, Yu; Wang, Hui; Yuan, Zhiyong

2018-04-19

Accurately identifying and quantifying cellular autophagy is very important as the significance of autophagy in physiological and pathological processes becomes increasingly evident. Ectopically expressed fluorescent-tagged microtubule-associated protein light chain 3B (MAP1LC3B, LC3) is the most widely used reporter for monitoring autophagy activity thus far. However, this approach ignores the influence of constitutively overexpressed LC3 on autophagy itself and autophagy-related processes and its accuracy in indicating autophagy is questionable. Here, we generated a knock-in GFP-LC3 reporter via the CRISPR/Cas9 system in 293FT cells to add GFP to the N-terminal of and in frame with endogenous LC3. We proved that this knock-in GFP-LC3 was expressed at biological level driven by the endogenous transcriptional regulatory elements as the wild type alleles. Compared with the ectopically expressed GFP-LC3, the endogenous knock-in reporter exhibited much higher sensitivity and signal-to-noise ratio of GFP-LC3 puncta upon the induction or inhibition of autophagy at certain step for monitoring autophagy activity. Thus, according to the previous reported concerning and the results presented here, we suggest that this knock-in GFP-LC3 reporter is better for bona fide monitoring cellular autophagy and should be employed for further study of autophagy in vitro and in vivo. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The 29-kDa proteins phosphorylated ion thrombin-activated human platelets are forms of the estrogen receptor-related 27-kDa heat shock protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mendelsohn, M.E.; Yan Zhu; O'Neill, S.

Thrombin plays a critical role in platelet activation, hemostasis, and thrombosis. Cellular activation by thrombin leads to the phosphorylation of multiple proteins, most of which are unidentified. The authors have characterized several 29-kDa proteins that are rapidly phosphorylated following exposure of intact human platelets to thrombin. A murine monoclonal antibody raised to an unidentified estrogen receptor-related 29-kDa protein selectively recognized these proteins as well as a more basic, unphosphorylated 27-kDa protein. Cellular activation by thrombin led to a marked shift in the proportion of protein from the 27-kDa unphosphorylated form to the 29-kDa phosphoprotein species. Using this antibody, they isolatedmore » and sequenced a human cDNA clone encoding a protein that was identical to the mammalian 27-kDa heat shock protein (HSP27), a protein of uncertain function that is known to be phosphorylated to several forms and to be transcriptionally induced by estrogen. The 29-kDa proteins were confirmed to be phosphorylated forms of HSP27 by immunoprecipitation studies. Thus, the estrogen receptor-related protein is HSP27, and the three major 20-kDa proteins phosphorylated in thrombin-activated platelets are forms of HSP27. These data suggest a role for HSP27 in the signal transduction events of platelet activation.« less

High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy.

PubMed

Bravo-San Pedro, J M; Pietrocola, F; Sica, V; Izzo, V; Sauvat, A; Kepp, O; Maiuri, M C; Kroemer, G; Galluzzi, L

2017-01-01

Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned organelle commonly known as autophagosome. Various methods have been developed to quantify the size of the autophagosomal compartment, which is an indirect indicator of macroautophagic responses, based on the peculiar ability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; best known as LC3) to accumulate in forming autophagosomes upon maturation. One particularly convenient method to monitor the accumulation of mature LC3 within autophagosomes relies on a green fluorescent protein (GFP)-tagged variant of this protein and fluorescence microscopy. In physiological conditions, cells transfected temporarily or stably with a GFP-LC3-encoding construct exhibit a diffuse green fluorescence over the cytoplasm and nucleus. Conversely, in response to macroautophagy-promoting stimuli, the GFP-LC3 signal becomes punctate and often (but not always) predominantly cytoplasmic. The accumulation of GFP-LC3 in cytoplasmic dots, however, also ensues the blockage of any of the steps that ensure the degradation of mature autophagosomes, calling for the implementation of strategies that accurately discriminate between an increase in autophagic flux and an arrest in autophagic degradation. Various cell lines have been engineered to stably express GFP-LC3, which-combined with the appropriate controls of flux, high-throughput imaging stations, and automated image analysis-offer a relatively straightforward tool to screen large chemical or biological libraries for inducers or inhibitors of autophagy. Here, we describe a simple and robust method for the high-throughput quantification of GFP-LC3 + dots by automated fluorescence microscopy. © 2017 Elsevier Inc. All rights reserved.

Expression of Anthocyanins and Proanthocyanidins after Transformation of Alfalfa with Maize Lc12

PubMed Central

Ray, Heather; Yu, Min; Auser, Patricia; Blahut-Beatty, Laureen; McKersie, Brian; Bowley, Steve; Westcott, Neil; Coulman, Bruce; Lloyd, Alan; Gruber, Margaret Y.

2003-01-01

Three anthocyanin regulatory genes of maize (Zea mays; Lc, B-Peru, and C1) were introduced into alfalfa (Medicago sativa) in a strategy designed to stimulate the flavonoid pathway and alter the composition of flavonoids produced in forage. Lc constructs included a full-length gene and a gene with a shortened 5′-untranslated region. Lc RNA was strongly expressed in Lc transgenic alfalfa foliage, but accumulation of red-purple anthocyanin was observed only under conditions of high light intensity or low temperature. These stress conditions induced chalcone synthase and flavanone 3-hydroxylase expression in Lc transgenic alfalfa foliage compared with non-transformed plants. Genotypes containing the Lc transgene construct with a full-length 5′-untranslated region responded more quickly to stress conditions and with a more extreme phenotype. High-performance liquid chromatography analysis of field-grown tissue indicated that flavone content was reduced in forage of the Lc transgenic plants. Leucocyanidin reductase, the enzyme that controls entry of metabolites into the proanthocyanidin pathway, was activated both in foliage and in developing seeds of the Lc transgenic alfalfa genotypes. Proanthocyanidin polymer was accumulated in the forage, but (+)-catechin monomers were not detected. B-Peru transgenic and C1 transgenic populations displayed no visible phenotypic changes, although these transgenes were expressed at detectable levels. These results support the emerging picture of Lc transgene-specific patterns of expression in different recipient species. These results demonstrate that proanthocyanidin biosynthesis can be stimulated in alfalfa forage using an myc-like transgene, and they pave the way for the development of high quality, bloat-safe cultivars with ruminal protein bypass. PMID:12857826

Diffuse Peritoneal and Bowel Wall Infiltration by Light Chain-AL Amyloidosis with Omental Calcification Mimicking Abdominal Carcinomatosis - An Elderly Female with Incidental Finding of Light Chain Monoclonal Gammopathy of Undetermined Significance (LC-MGUS).

PubMed

Junejo, Shoaib; Ali, Yasir; Singh Lubana, Sandeep; Tuli, Sandeep S

2017-11-25

BACKGROUND Amyloidosis is the extracellular tissue deposition of plasma proteins, which after conformational changes, forms antiparallel beta pleated sheets of fibrils. Amyloid light-chain (AL) is a type of amyloidosis that is due to deposition of proteins derived from immunoglobulin (Ig) light chains. Gastrointestinal tract (GIT) involvement most often found in amyloid A (AA) amyloidosis type. There have been no reports of obstructive GIT AL amyloid patients having monoclonal gammopathy of undetermined significance (MGUS). Our case is the first case to show two coinciding conditions; one is the association of GIT AL amyloidosis with the incidental finding of a rare type of MGUS (LC-MGUS) and the other is the radiologic presentation of GIT amyloidosis with omental calcification mimicking the GIT malignancy. CASE REPORT A 68-year-old female presented with symptoms of partial bowel obstruction, including intermittent diffuse abdominal pain and constipation. After computed tomography (CT) abdomen and pelvis, an exploratory laparotomy was needed because of suspicion of abdominal carcinomatosis due to diffuse omental calcification. The tissue sent for biopsy surprisingly showed AL amyloidosis. The patient did not report any systemic symptoms. Further workup was advised to inquire about the plasma cell dyscrasia which eventually turned into a very rare version of MGUS knows as light chain MGUS (LC-MGUS). Following adequate resection of the involved structures, the patient was then placed on chemotherapy and successfully went into remission. CONCLUSIONS This case report illustrates that in an era of evidence based medicine, it is important to show through case reports the association of GIT AL amyloidosis with LC-MGUS, as the literature on this topic is lacking. It also points to the importance of timely intervention that can greatly enhance, not only the only the chances of remission but also prevention of further complications such as malignant transformation.

Light-induced vegetative anthocyanin pigmentation in Petunia

PubMed Central

Albert, Nick W.; Lewis, David H.; Zhang, Huaibi; Irving, Louis J.; Jameson, Paula E.; Davies, Kevin M.

2009-01-01

The Lc petunia system, which displays enhanced, light-induced vegetative pigmentation, was used to investigate how high light affects anthocyanin biosynthesis, and to assess the effects of anthocyanin pigmentation upon photosynthesis. Lc petunia plants displayed intense purple anthocyanin pigmentation throughout the leaves and stems when grown under high-light conditions, yet remain acyanic when grown under shade conditions. The coloured phenotypes matched with an accumulation of anthocyanins and flavonols, as well as the activation of the early and late flavonoid biosynthetic genes required for flavonol and anthocyanin production. Pigmentation in Lc petunia only occurred under conditions which normally induce a modest amount of anthocyanin to accumulate in wild-type Mitchell petunia [Petunia axillaris×(Petunia axillaris×Petunia hybrida cv. ‘Rose of Heaven’)]. Anthocyanin pigmentation in Lc petunia leaves appears to screen underlying photosynthetic tissues, increasing light saturation and light compensation points, without reducing the maximal photosynthetic assimilation rate (Amax). In the Lc petunia system, where the bHLH factor Leaf colour is constitutively expressed, expression of the bHLH (Lc) and WD40 (An11) components of the anthocyanin regulatory system were not limited, suggesting that the high-light-induced anthocyanin pigmentation is regulated by endogenous MYB transcription factors. PMID:19380423

Measurement of Ether Phospholipids in Human Plasma with HPLC-ELSD and LC/ESI-MS After Hydrolysis of Plasma with Phospholipase A1.

PubMed

Mawatari, Shiro; Hazeyama, Seira; Fujino, Takehiko

2016-08-01

Ethanolamine ether phospholipid (eEtnGpl) and choline ether phospholipid (eChoGpl) are present in human plasma or serum, but the relative concentration of the ether phospholipids in plasma is very low as compared to those in other tissues. Nowadays, measurement of ether phospholipids in plasma depends on tandem mass spectrometry (LC/MS/MS), but a system for LC/MS/MS is generally too expensive for usual clinical laboratories. Treatment of plasma with phospholipase A1 (PLA1) causes complete hydrolysis of diacylphospholipids, but ether phospholipids remain intact. After the treatment of plasma with PLA1, both eEtnGpl and eChoGpl are detected as independent peaks by high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD). The same sample used for HPLC-ELSD can be applied to detect eEtnGpl and eChoGpl with electrospray ionization mass spectrometry. Presence of alkylacylphospholipids in both eChoGpl and eEtnGpl in human plasma was indicated by sequential hydrolysis of plasma with PLA1 and hydrochloric acid.

AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1

PubMed Central

Strappazzon, F; Nazio, F; Corrado, M; Cianfanelli, V; Romagnoli, A; Fimia, G M; Campello, S; Nardacci, R; Piacentini, M; Campanella, M; Cecconi, F

2015-01-01

Damaged mitochondria are eliminated by mitophagy, a selective form of autophagy whose dysfunction associates with neurodegenerative diseases. PINK1, PARKIN and p62/SQTMS1 have been shown to regulate mitophagy, leaving hitherto ill-defined the contribution by key players in ‘general' autophagy. In basal conditions, a pool of AMBRA1 – an upstream autophagy regulator and a PARKIN interactor – is present at the mitochondria, where its pro-autophagic activity is inhibited by Bcl-2. Here we show that, upon mitophagy induction, AMBRA1 binds the autophagosome adapter LC3 through a LIR (LC3 interacting region) motif, this interaction being crucial for regulating both canonical PARKIN-dependent and -independent mitochondrial clearance. Moreover, forcing AMBRA1 localization to the outer mitochondrial membrane unleashes a massive PARKIN- and p62-independent but LC3-dependent mitophagy. These results highlight a novel role for AMBRA1 as a powerful mitophagy regulator, through both canonical or noncanonical pathways. PMID:25215947

AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1.

PubMed

Strappazzon, F; Nazio, F; Corrado, M; Cianfanelli, V; Romagnoli, A; Fimia, G M; Campello, S; Nardacci, R; Piacentini, M; Campanella, M; Cecconi, F

2015-03-01

Damaged mitochondria are eliminated by mitophagy, a selective form of autophagy whose dysfunction associates with neurodegenerative diseases. PINK1, PARKIN and p62/SQTMS1 have been shown to regulate mitophagy, leaving hitherto ill-defined the contribution by key players in 'general' autophagy. In basal conditions, a pool of AMBRA1 - an upstream autophagy regulator and a PARKIN interactor - is present at the mitochondria, where its pro-autophagic activity is inhibited by Bcl-2. Here we show that, upon mitophagy induction, AMBRA1 binds the autophagosome adapter LC3 through a LIR (LC3 interacting region) motif, this interaction being crucial for regulating both canonical PARKIN-dependent and -independent mitochondrial clearance. Moreover, forcing AMBRA1 localization to the outer mitochondrial membrane unleashes a massive PARKIN- and p62-independent but LC3-dependent mitophagy. These results highlight a novel role for AMBRA1 as a powerful mitophagy regulator, through both canonical or noncanonical pathways.

Lipin-1 regulates Bnip3-mediated mitophagy in glycolytic muscle.

PubMed

Alshudukhi, Abdullah A; Zhu, Jing; Huang, Dengtong; Jama, Abdulrahman; Smith, Jeffrey D; Wang, Qing Jun; Esser, Karyn A; Ren, Hongmei

2018-06-25

Autophagy of mitochondria (mitophagy) is essential for maintaining muscle mass and healthy skeletal muscle. Patients with heritable phosphatidic acid phosphatase lipin-1-null mutations present with severe rhabdomyolysis and muscle atrophy in glycolytic muscle fibers, which are accompanied with mitochondrial aggregates and reduced mitochondrial cytochrome c oxidase activity. However, the underlying mechanisms leading to muscle atrophy as a result of lipin-1 deficiency are still not clear. In this study, we found that lipin-1 deficiency in mice is associated with a marked accumulation of abnormal mitochondria and autophagic vacuoles in glycolytic muscle fibers. Our studies using lipin-1-deficient myoblasts suggest that lipin-1 participates in B-cell leukemia (BCL)-2 adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3)-regulated mitophagy by interacting with microtubule-associated protein 1A/1B-light chain (LC)3, which is an important step in the recruitment of mitochondria to nascent autophagosomes. The requirement of lipin-1 for Bnip3-mediated mitophagy was further verified in vivo in lipin-1-deficient green fluorescent protein-LC3 transgenic mice (lipin-1 -/- -GFP-LC3). Finally, we showed that lipin-1 deficiency in mice resulted in defective mitochondrial adaptation to starvation-induced metabolic stress and impaired contractile muscle force in glycolytic muscle fibers. In summary, our study suggests that deregulated mitophagy arising from lipin-1 deficiency is associated with impaired muscle function and may contribute to muscle rhabdomyolysis in humans.-Alshudukhi, A. A., Zhu, J., Huang, D., Jama, A., Smith, J. D., Wang, Q. J., Esser, K. A., Ren, H. Lipin-1 regulates Bnip3-mediated mitophagy in glycolytic muscle.

Lattice-patterned LC-polymer composites containing various nanoparticles as additives

PubMed Central

2012-01-01

In this study, we show the effect of various nanoparticle additives on phase separation behavior of a lattice-patterned liquid crystal [LC]-polymer composite system and on interfacial properties between the LC and polymer. Lattice-patterned LC-polymer composites were fabricated by exposing to UV light a mixture of a prepolymer, an LC, and SiO2 nanoparticles positioned under a patterned photomask. This resulted in the formation of an LC and prepolymer region through phase separation. We found that the incorporation of SiO2 nanoparticles significantly affected the electro-optical properties of the lattice-patterned LC-polymer composites. This effect is a fundamental characteristic of flexible displays. The electro-optical properties depend on the size and surface functional groups of the SiO2 nanoparticles. Compared with untreated pristine SiO2 nanoparticles, which adversely affect the performance of LC molecules surrounded by polymer walls, SiO2 nanoparticles with surface functional groups were found to improve the electro-optical properties of the lattice-patterned LC-polymer composites by increasing the quantity of SiO2 nanoparticles. The surface functional groups of the SiO2 nanoparticles were closely related to the distribution of SiO2 nanoparticles in the LC-polymer composites, and they influenced the electro-optical properties of the LC molecules. It is clear from our work that the introduction of nanoparticles into a lattice-patterned LC-polymer composite provides a method for controlling and improving the composite's electro-optical properties. This technique can be used to produce flexible substrates for various flexible electronic devices. PMID:22222011

Antibody to liver cytosol (anti-LC1) in patients with autoimmune chronic active hepatitis type 2.

PubMed

Martini, E; Abuaf, N; Cavalli, F; Durand, V; Johanet, C; Homberg, J C

1988-01-01

A new autoantibody was detected by immunoprecipitation in the serum of 21 patients with chronic active hepatitis. The antibody reacted against a soluble cytosolic antigen in liver. The antibody was organ specific but not species specific and was therefore called anti-liver cytosol antibody Type 1 (anti-LC1). In seven of 21 cases, no other autoantibody was found; the remaining 14 cases had anti-liver/kidney microsome antibody Type 1 (anti-LKM1). With indirect immunofluorescence, a distinctive staining pattern was observed with the seven sera with anti-LC1 and without anti-LKM1. The antibody stained the cytoplasm of hepatocytes from four different animal species and spared the cellular layer around the central veins of mouse and rat liver that we have called juxtavenous hepatocytes. The immunofluorescence pattern disappeared after absorption of sera by a liver cytosol fraction. The 14 sera with both antibodies displayed anti-LC1 immunofluorescent pattern after absorption of anti-LKM1 by the liver microsomal fraction. The anti-LC1 was found in the serum only in patients with chronic active hepatitis of unknown cause. Anti-LC1 antibody was not found in sera from 100 patients with chronic active hepatitis associated with anti-actin antibody classic chronic active hepatitis Type 1, 100 patients with primary biliary cirrhosis, 157 patients with drug-induced hepatitis and a large number of patients with liver and nonliver diseases. This new antibody was considered a second marker of chronic active hepatitis associated with anti-LKM1 (anti-LKM1 chronic active hepatitis) or autoimmune chronic active hepatitis Type 2.

Cloning, expression and activation of a truncated 92-kDa gelatinase minienzyme.

PubMed

Kröger, M; Tschesche, H

1997-09-01

The matrix metalloproteinases (MMPs) are a family of highly homologous zinc-endopeptidases that degrade extracellular matrix components. Human 92-kDa gelatinase (MMP-9) represents one of the MMPs that cleaves native collagen type IV. As a basis for structural investigations, the short form (catalytic domain, amino acid residues 113-450) of the 92-kDa gelatinase cDNA was cloned and expressed in E. coli as a minienzyme. By combination of reverse transcription (RT) and polymerase chain reaction (PCR), the truncated 92-kDa gelatinase-cDNA was amplified from the corresponding mRNA derived from ovarian carcinoma cells. The cDNA fragment obtained was cloned in E. coli and sequenced. With the exception of one nucleotide inversion at position 745 (gt-->tg) the cDNA sequence was identical to the nucleotide sequence of the 92-kDa gelatinase as has been previously reported. The protein was expressed in E. coli using the vector pET-12b. The recombinant protein was stored in inclusion bodies and extracted as a 38 kDa species from the inclusion bodies by solubilization in 8 M urea. The product was purified by affinity chromatography and gel filtration. Amino-terminal sequence analysis confirmed the identity with the catalytic domain of 92-kDa gelatinase. The recombinant protein was refolded in the presence of Ca2+ and Zn2+ and yielded an active minienzyme with gelatinolytic activity. It degrades the native substrate collagen type IV and the synthetic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 x AcOH like the full-length 92-kDa gelatinase. The catalytic activity could be inhibited by the specific MMP inhibitors TIMP-1 and TIMP-2.

Recombinant expression, isolation, and proteolysis of extracellular matrix-secreted phosphoprotein-24 kDa.

PubMed

Murray, Elsa J Brochmann; Murray, Samuel S; Simon, Robert; Behnam, Keyvan

2007-01-01

Secreted phosphoprotein-24 kDa (spp24) is an extracellular matrix protein first cloned from bone. Bovine spp24 is transcribed as a 203 amino acid residue protein that undergoes cleavage of a secretory peptide to form the mature protein (spp24, residues 24 to 203). While not osteogenic itself, spp24 is degraded to a pro-osteogenic protein, spp18.5, in bone. Both spp18.5 and spp24 contain a cyclic TRH1 (TGF-beta receptor II homology-1) domain similar to that found in the receptor itself and in fetuin. A synthetic peptide corresponding to the TRH1 domain of spp18.5 and spp24 specifically binds BMP-2 and enhances the rate and magnitude of BMP-2-induced ectopic bone formation in vivo. The parental protein, spp24, exhibits a high affinity for bone and mineral complexes, but its abundance there is low, suggesting that it is rapidly degraded. The availability of recombinant spp24 and its degradation products would facilitate the elucidation of their structure:function relationships. We describe here the expression of His(6)-tagged bovine spp24 (residues 24 to 203) in E. coli, its purification by high-resolution IMAC (immobilized metal affinity chromatography), and the characterization of the full-length recombinant 21.5 kDa protein and its two major 16 kDa and 14.5 kDa degradation products (spp24, residues 24 to 157, and spp24, residues 24 to 143) by mass spectroscopy. The recombinant spp24 protein was resistant to proteolysis by MC3T3-E1 osteoblastic cell extracts in the absence of calcium; however, in the presence of 4 mM Ca, it can undergo essentially complete proteolysis to small peptides, bypassing the 16 kDa and 14.5 kDa intermediates. This confirms the proteolytic susceptibility of spp24. It also suggests that the levels of spp24 in bone may be regulated, in part, by calcium-dependent proteolysis mediated by osteoblastic cells.

Bisphenol A release from an orthodontic resin composite: A GC/MS and LC/MS study.

PubMed

Deviot, Marc; Lachaise, Isabelle; Högg, Christof; Durner, Jürgen; Reichl, Franz-Xaver; Attal, Jean-Pierre; Dursun, Elisabeth

2018-02-01

First, to analyse the in vitro release of BPA and Bis-GMA from an orthodontic resin composite (Transbond XT, 3M Unitek), stored in various conditions, by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS); then to extrapolate the data to the clinical situation. Secondly, to explore the thermal stability of Bis-GMA. Cylinders of resin composite were prepared and stored according to 3 different protocols: (1) they were light-cured 20s, then placed in artificial saliva; (2) they were light-cured 2s, then placed in acetonitrile; (3) they were light-cured 2s, then placed in methanol. For each group, BPA and Bis-GMA release were determined with GC/MS and/or LC/MS at least after one week. Besides, 120 brackets (10 of each type) were bonded over metal teeth, then debonded, and the weight and the surface of resin composite residues were measured. BPA and Bis-GMA release of adhesive residues were extrapolated from the data obtained with the cylinders. Besides, BPA release from a heated Bis-GMA solution was measured. With GC/MC, BPA was detected in all samples. With LC/MS, BPA was detected only from samples immersed in MeOH; Bis-GMA was detected, in varying amount according to the extraction media and the light-curing time. BPA was found after heating of the Bis-GMA solution. Contamination risk and the heat applied in GC/MS may overestimate the BPA release from resin composite. Based on the LC/MS results, the risk of BPA release after orthodontic bonding would be more than 42000 times lower than the TDI for a 30-kg child. Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

S100A8 as potential salivary biomarker of oral squamous cell carcinoma using nanoLC-MS/MS.

PubMed

Jou, Yu-Jen; Hua, Chun-Hung; Lin, Chia-Der; Lai, Chih-Ho; Huang, Su-Hua; Tsai, Ming-Hsui; Kao, Jung-Yie; Lin, Cheng-Wen

2014-09-25

Oral squamous cell carcinoma (OSCC) shows low 5-year survival; early treatment greatly reduces mortality and morbidity. Saliva is a non-invasive sample, with good potential to discover biomarkers for early detection. NanoLC-MS/MS served to analyze saliva proteome from control subjects (n=35) and OSCC patients T1 (n=29), T2 (n=36), T3 (n=14) and T4 (n=21) stages. Identified biomarkers were verified by Western blot and ELISA assays. NanoLC-MS/MS analysis of salivary proteins between 10 and 15kDa identified S100A8, hemoglobin delta and gamma-G globin in T3 and T4 stage OSCC as well as S100A7 in T1 and T2 stage OSCC. Western blot and ELISA indicated positive correlation between salivary S100A8 increment and tumor size stage. High level of S100A8 appeared in 3.4, 13.9, 92.9, and 100% of saliva OSCC patients with T1, T2, T3, and T4 stages, respectively. Significant increase of salivary S100A7 was observed in 20.7% and 11.1% of those with T1 and T2, respectively. AUROC curve indicated high sensitivity, specificity and accuracy of S100A8-based ELISA as a detector. NanoLC-MS/MS, Western blot and ELISA manifested salivary S100A8 as a specific and sensitive marker for detection of OSCC patients. Salivary S100A8 protein could be applicable in developing OSCC diagnostics. Copyright © 2014 Elsevier B.V. All rights reserved.

Spatiotemporal alterations of autophagy marker LC3 in rat skin fibroblasts during wound healing process

PubMed Central

Asai, Emiko; Yamamoto, Masaya; Ueda, Kazuki; Waguri, Satoshi

2018-01-01

Abstract To investigate the possible implications of autophagy, one of the degradation pathways induced by metabolic stress, in the dynamic reconstructive process of wound healing, the appearance and changes of punctate structures for microtubule-associated protein 1 light chain 3 (LC3), an autophagosome marker, were examined in a rat skin wound healing model. Although the ratio of LC3-II/LC3-I in Western blotting was not evidently changed during the wound healing process, LC3-positive dots were clearly observed in fibroblasts and myofibroblasts, and occasionally in macrophages, by immunohistofluorescence microscopy. Some of the LC3-positive dots were colocalized with Atg16L signal, an isolation membrane marker, and electron microscopy revealed the presence of typical autophagosomes in fibroblasts near the margin of the wound. The number of LC3-positive dots per fibroblast increased during the later period of the proliferation phase, and interestingly, it was higher in the margin than the center of the wound. It was also high in the periwound skin area. These results suggest that drastic functional changes in fibroblasts during wound healing process are accompanied by the alteration of the autophagy-lysosomal degradation system. PMID:29343655

Spatiotemporal alterations of autophagy marker LC3 in rat skin fibroblasts during wound healing process.

PubMed

Asai, Emiko; Yamamoto, Masaya; Ueda, Kazuki; Waguri, Satoshi

2018-04-17

To investigate the possible implications of autophagy, one of the degradation pathways induced by metabolic stress, in the dynamic reconstructive process of wound healing, the appearance and changes of punctate structures for microtubule-associated protein 1 light chain 3 (LC3), an autophagosome marker, were examined in a rat skin wound healing model. Although the ratio of LC3-II/LC3-I in Western blotting was not evidently changed during the wound healing process, LC3-positive dots were clearly observed in fibroblasts and myofibroblasts, and occasionally in macrophages, by immunohistofluorescence microscopy. Some of the LC3-positive dots were colocalized with Atg16L signal, an isolation membrane marker, and electron microscopy revealed the presence of typical autophagosomes in fibroblasts near the margin of the wound. The number of LC3-positive dots per fibroblast increased during the later period of the proliferation phase, and interestingly, it was higher in the margin than the center of the wound. It was also high in the periwound skin area. These results suggest that drastic functional changes in fibroblasts during wound healing process are accompanied by the alteration of the autophagy-lysosomal degradation system.

Modeling and Docking Studies on Novel Mutants (K71L and T204V) of the ATPase Domain of Human Heat Shock 70 kDa Protein 1

PubMed Central

Elengoe, Asita; Naser, Mohammed Abu; Hamdan, Salehhuddin

2014-01-01

The purpose of exploring protein interactions between human adenovirus and heat shock protein 70 is to exploit a potentially synergistic interaction to enhance anti-tumoral efficacy and decrease toxicity in cancer treatment. However, the protein interaction of Hsp70 with E1A32 kDa of human adenovirus serotype 5 remains to be elucidated. In this study, two residues of ATPase domain of human heat shock 70 kDa protein 1 (PDB: 1 HJO) were mutated. 3D mutant models (K71L and T204V) using PyMol software were then constructed. The structures were evaluated by PROCHECK, ProQ, ERRAT, Verify 3D and ProSA modules. All evidence suggests that all protein models are acceptable and of good quality. The E1A32 kDa motif was retrieved from UniProt (P03255), as well as subjected to docking interaction with NBD, K71L and T204V, using the Autodock 4.2 program. The best lowest binding energy value of −9.09 kcal/mol was selected for novel T204V. Moreover, the protein-ligand complex structures were validated by RMSD, RMSF, hydrogen bonds and salt bridge analysis. This revealed that the T204V-E1A32 kDa motif complex was the most stable among all three complex structures. This study provides information about the interaction between Hsp70 and the E1A32 kDa motif, which emphasizes future perspectives to design rational drugs and vaccines in cancer therapy. PMID:24758925

Structural features of LC8-induced self-association of swallow.

PubMed

Kidane, Ariam I; Song, Yujuan; Nyarko, Afua; Hall, Justin; Hare, Michael; Löhr, Frank; Barbar, Elisar

2013-09-03

Cell functions depend on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than that of dynein, and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the energetics and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, intermonomer nuclear Overhauser effects assigned to residues expected at interface positions, and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled coil. Secondary chemical shifts and (15)N backbone relaxation identify the boundaries and distinguishing structural features of the coiled coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled coil is critical for bicoid mRNA localization because it favors structural organization of Swallow, which except for the central LC8-promoted coiled coil is primarily disordered.

Structural Features of LC8-Induced Self Association of Swallow†

PubMed Central

Kidane, Ariam I.; Song, Yujuan; Nyarko, Afua; Hall, Justin; Hare, Michael; Löhr, Frank; Barbar, Elisar

2013-01-01

Cell function depends on the collective activity of protein networks within which a few proteins, called hubs, participate in a large number of interactions. Dynein light chain LC8, first discovered as a subunit of the motor protein dynein, is considered to have a role broader than dynein and its participation in diverse systems fits the description of a hub. Among its partners is Swallow with which LC8 is essential for proper localization of bicoid mRNA at the anterior cortex of Drosophila oocytes. Why LC8 is essential in this process is not clear, but emerging evidence suggests that LC8 functions by promoting self-association and/or structural organization of its diverse binding partners. This work addresses the mechanistic and structural features of LC8-induced Swallow self-association distant from LC8 binding. Mutational design based on a hypothetical helical wheel, inter-monomer NOEs assigned to residues expected at interface positions and circular dichroism spectral characteristics indicate that the LC8-promoted dimer of Swallow is a coiled-coil. Secondary chemical shifts and 15N backbone relaxation identify the boundaries and distinguishing structural features of the coiled-coil. Thermodynamic analysis of Swallow polypeptides designed to decouple self-association from LC8 binding reveals that the higher binding affinity of the engineered bivalent Swallow is of purely entropic origin and that the linker separating the coiled-coil from the LC8 binding site remains disordered. We speculate that the LC8-promoted coiled-coil is critical for bicoid mRNA localization because it could induce structural organization of Swallow, which except for the central LC8-promoted coiled-coil is primarily disordered. PMID:23914803

Liquid crystal light valve technologies for display applications

NASA Astrophysics Data System (ADS)

Kikuchi, Hiroshi; Takizawa, Kuniharu

2001-11-01

The liquid crystal (LC) light valve, which is a spatial light modulator that uses LC material, is a very important device in the area of display development, image processing, optical computing, holograms, etc. In particular, there have been dramatic developments in the past few years in the application of the LC light valve to projectors and other display technologies. Various LC operating modes have been developed, including thin film transistors, MOS-FETs and other active matrix drive techniques to meet the requirements for higher resolution, and substantial improvements have been achieved in the performance of optical systems, resulting in brighter display images. Given this background, the number of applications for the LC light valve has greatly increased. The resolution has increased from QVGA (320 x 240) to QXGA (2048 x 1536) or even super- high resolution of eight million pixels. In the area of optical output, projectors of 600 to 13,000 lm are now available, and they are used for presentations, home theatres, electronic cinema and other diverse applications. Projectors using the LC light valve can display high- resolution images on large screens. They are now expected to be developed further as part of hyper-reality visual systems. This paper provides an overview of the needs for large-screen displays, human factors related to visual effects, the way in which LC light valves are applied to projectors, improvements in moving picture quality, and the results of the latest studies that have been made to increase the quality of images and moving images or pictures.

Isoform composition and stoichiometry of the approx. 90-kDa heat shock protein associated with glucocorticoid receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mendel, D.B.; Orti, E.

1988-05-15

The authors observed that the approx. 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approx. 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approx. 90-kDa heat shock protein. The observation that TSTA and the approx. 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested thatmore » the doublet observed is also due to the existence of two isoforms. They have therefore conducted this study to determine whether TSTA and the approx. 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approx. 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. They used the BuGr1 and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approx. 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with (/sup 35/S)methionine to metabolically label proteins to steady state. The long-term metabolic labeling approach has also enabled them to directly determine that the purified non-activated glucocorticoid receptor contains a single steroid-binding protein and two approx. 90-kDa non-steroid-binding subunits. The consistency with which a approx. 1:2 stoichiometric ratio of steroid binding to approx. 90-kDa protein is observed supports the view that the approx. 90-kDa heat shock protein is a true component of nonactivated glucocorticoid-receptor complexes.« less

The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

PubMed

Manolson, M F; Proteau, D; Preston, R A; Stenbit, A; Roberts, B T; Hoyt, M A; Preuss, D; Mulholland, J; Botstein, D; Jones, E W

1992-07-15

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.

Myosins and DYNLL1/LC8 in the honey bee (Apis mellifera L.) brain.

PubMed

Calábria, Luciana Karen; Peixoto, Pablo Marco Veras; Passos Lima, Andreia Barcelos; Peixoto, Leonardo Gomes; de Moraes, Viviane Rodrigues Alves; Teixeira, Renata Roland; Dos Santos, Claudia Tavares; E Silva, Letícia Oliveira; da Silva, Maria de Fátima Rodrigues; dos Santos, Ana Alice Diniz; Garcia-Cairasco, Norberto; Martins, Antônio Roberto; Espreafico, Enilza Maria; Espindola, Foued Salmen

2011-09-01

Honey bees have brain structures with specialized and developed systems of communication that account for memory, learning capacity and behavioral organization with a set of genes homologous to vertebrate genes. Many microtubule- and actin-based molecular motors are involved in axonal/dendritic transport. Myosin-Va is present in the honey bee Apis mellifera nervous system of the larvae and adult castes and subcastes. DYNLL1/LC8 and myosin-IIb, -VI and -IXb have also been detected in the adult brain. SNARE proteins, such as CaMKII, clathrin, syntaxin, SNAP25, munc18, synaptophysin and synaptotagmin, are also expressed in the honey bee brain. Honey bee myosin-Va displayed ATP-dependent solubility and was associated with DYNLL1/LC8 and SNARE proteins in the membrane vesicle-enriched fraction. Myosin-Va expression was also decreased after the intracerebral injection of melittin and NMDA. The immunolocalization of myosin-Va and -IV, DYNLL1/LC8, and synaptophysin in mushroom bodies, and optical and antennal lobes was compared with the brain morphology based on Neo-Timm histochemistry and revealed a distinct and punctate distribution. This result suggested that the pattern of localization is associated with neuron function. Therefore, our data indicated that the roles of myosins, DYNLL1/LC8, and SNARE proteins in the nervous and visual systems of honey bees should be further studied under different developmental, caste and behavioral conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Choline dehydrogenase interacts with SQSTM1/p62 to recruit LC3 and stimulate mitophagy.

PubMed

Park, Sungwoo; Choi, Seon-Guk; Yoo, Seung-Min; Son, Jin H; Jung, Yong-Keun

2014-01-01

CHDH (choline dehydrogenase) is an enzyme catalyzing the dehydrogenation of choline to betaine aldehyde in mitochondria. Apart from this well-known activity, we report here a pivotal role of CHDH in mitophagy. Knockdown of CHDH expression impairs CCCP-induced mitophagy and PARK2/parkin-mediated clearance of mitochondria in mammalian cells, including HeLa cells and SN4741 dopaminergic neuronal cells. Conversely, overexpression of CHDH accelerates PARK2-mediated mitophagy. CHDH is found on both the outer and inner membranes of mitochondria in resting cells. Interestingly, upon induction of mitophagy, CHDH accumulates on the outer membrane in a mitochondrial potential-dependent manner. We found that CHDH is not a substrate of PARK2 but interacts with SQSTM1 independently of PARK2 to recruit SQSTM1 into depolarized mitochondria. The FB1 domain of CHDH is exposed to the cytosol and is required for the interaction with SQSTM1, and overexpression of the FB1 domain only in cytosol reduces CCCP-induced mitochondrial degradation via competitive interaction with SQSTM1. In addition, CHDH, but not the CHDH FB1 deletion mutant, forms a ternary protein complex with SQSTM1 and MAP1LC3 (LC3), leading to loading of LC3 onto the damaged mitochondria via SQSTM1. Further, CHDH is crucial to the mitophagy induced by MPP+ in SN4741 cells. Overall, our results suggest that CHDH is required for PARK2-mediated mitophagy for the recruitment of SQSTM1 and LC3 onto the mitochondria for cargo recognition.

Identification by GeLC-MS/MS of trypsin inhibitor in sarcoplasmic proteins of three tropical fish and characterization of their inhibitory properties.

PubMed

Siriangkanakun, Siriphon; Li-Chan, Eunice C Y; Yongsawadigul, Jirawat

2014-07-01

Sarcoplasmic proteins from 3 fish species were fractionated by 50% to 70% ammonium sulfate precipitation. Lyophilized fractionated sarcoplasmic proteins of threadfin bream (TB-SP), bigeye snapper (BS-SP), and yellow croaker (YC-SP) showed 80% to 92% trypsin inhibitory activity. Trypsin inhibitory activity staining gel electrophoresis revealed bands at 32, 33, 37, 45, 48, and 50 kDa for the 3 species, and a band at 95 kDa was observed for TB-SP and YC-SP. Alpha-1-antitrypsin with molecular mass of 45 to 50 kDa was identified in YC-SP by gel-based liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Other major protein bands appeared on trypsin activity staining included phosphorylase, glyceraldehyde-3-phosphate dehydrogenase, and creatine kinase with molecular mass of 95 and 35 to 40 kDa, respectively. But, these 3 proteins did not show true trypsin inhibitory activity. Trypsin inhibitory activity of fractionated sarcoplasmic proteins showed good stability, with >80% activity retained at 60 °C and up to 0.6 M NaCl. TB-SP showed the highest inhibitory activity against autolysis of washed threadfin bream mince at 65 °C. Addition of 0.5% or 1% TB-SP improved textural properties of threadfin bream surimi gels preincubated at 37 or 65 °C followed by heating at 90 °C. Therefore, TB-SP could be a promising protein ingredient for enhancing surimi gel texture. Threadfin bream, bigeye snapper, and yellow croaker are the main species used as raw material for tropical surimi production. Sarcoplasmic proteins from 3 species contain trypsin inhibitor(s) that can minimize proteolytic activity and improve gel texture of proteinase-laden fish muscle. Therefore, sarcoplasmic proteins that are byproducts from surimi processing of these species could be recovered, fractionated, and utilized as a functional protein ingredient. © 2014 Institute of Food Technologists®

Characterization of photosystem 1 chlorophyll a/b-binding apoprotein accumulation in developing soybean using type-specific antibodies

NASA Technical Reports Server (NTRS)

Henry, R. L.; Armbrust, T.; Gallegos, G.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

The structure and supramolecular assembly of the soybean photosystem 1 (PS 1) chlorophyll a/b-binding antenna (LHC 1) was examined. We identified the subunit composition of LHC 1 in soybean and followed the accumulation of individual subunits during light-induced assembly. We observed four LHC 1 subunits, at 23, 22, 21 and 20.5 kDa, obtained partial sequence information by amino-terminal sequence analysis, and classified the 20.5, 22, and 21 kDa subunits as being encoded by type I, II, and IV chlorophyll a/b binding protein genes, respectively. Antisera against LHC 1 subunits were used to follow the accumulation of individual subunits during the light-initiated transition from etioplast to chloroplast. Several points are noteworthy. First, monospecific antibody against the 22 kDa subunit decorated a 25 kDa peptide in etiolated tissue, which declined during maturation. This decline correlated with the light-induced appearance of mature 22 kDa peptide, suggesting a precursor/product relationship. Second, the same antibody identified a 22 kDa protein in mature corn, but not a larger band in etiolated corn, suggesting that LHC 1 accumulation is regulated differently between species before the onset of chlorophyll biosynthesis. Third, the mature 22 kDa subunit appeared somewhat later than the other LHC 1 peptides during greening, implying that this subunit is less intimately associated with the PS1 core than are the subunits appearing earlier in development.

PEGylated-nanoliposomal clusterin for amyloidogenic light chain-induced endothelial dysfunction.

PubMed

Guzman-Villanueva, Diana; Migrino, Raymond Q; Truran, Seth; Karamanova, Nina; Franco, Daniel A; Burciu, Camelia; Senapati, Subhadip; Nedelkov, Dobrin; Hari, Parameswaran; Weissig, Volkmar

2018-06-01

Light chain (AL) amyloidosis is a disease associated with significant morbidity and mortality arising from multi-organ injury induced by amyloidogenic light chain proteins (LC). There is no available treatment to reverse the toxicity of LC. We previously showed that chaperone glycoprotein clusterin (CLU) and nanoliposomes (NL), separately, restore human microvascular endothelial function impaired by LC. In this work, we aim to prepare PEGylated-nanoliposomal clusterin (NL-CLU) formulations that could allow combined benefit against LC while potentially enabling efficient delivery to microvascular tissue, and test efficacy on human arteriole endothelial function. NL-CLU was prepared by a conjugation reaction between the carboxylated surface of NL and the primary amines of the CLU protein. NL were made of phosphatidylcholine (PC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG 2000 carboxylic acid) at 70:25:5 mol%. The protective effect of NL-CLU was tested by measuring the dilation response to acetylcholine and papaverine in human adipose arterioles exposed to LC. LC treatment significantly reduced the dilation response to acetylcholine and papaverine; co-treatment of LC with PEGylated-nanoliposomal CLU or free CLU restored the dilator response. NL-CLU is a feasible and promising approach to reverse LC-induced endothelial damage.

Autophagy-related proteins (p62, NBR1 and LC3) in intranuclear inclusions in neurodegenerative diseases.

PubMed

Mori, Fumiaki; Tanji, Kunikazu; Odagiri, Saori; Toyoshima, Yasuko; Yoshida, Mari; Kakita, Akiyoshi; Takahashi, Hitoshi; Wakabayashi, Koichi

2012-08-01

Incorporation of ubiquitin and ubiquitin-related proteins including p62 into neuronal intranuclear inclusions (NIIs) has been reported in a variety of neurodegenerative diseases. However, involvement of autophagy-specific proteins (NBR1 and LC3) in NIIs has not been mentioned. We immunohistochemically examined the brain of patients with Machado-Joseph disease (MJD; n=5), dentatorubral-pallidoluysian atrophy (DRPLA; n=5) and intranuclear inclusion body disease (INIBD; n=5), using antibodies against ubiquitin, p62, NBR1 and LC3. The proportion of p62-, NBR1- and LC3-positive inclusions relative to the number of ubiquitin-positive inclusions was calculated in each case. NIIs were positive for p62 in MJD (19.3%), DRPLA (49.7%) and INIBD (99.8%). As for autophagy-specific proteins, NIIs were positive for NBR1 in MJD (4.2%), DRPLA (5.5%) and INIBD (13.2%) and negative for LC3 in MJD, DRPLA and INIBD, except for one case of INIBD. These findings suggest that autophagy-lysosome pathway is not involved in the formation/degradation of NIIs. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Dynamic evolution of light-induced orientation of dye-doped liquid crystals in liquid phase studied by time-resolved optically heterodyned optical Kerr effect technique.

PubMed

Yang, Pei; Liu, Liying; Xu, Lei

2008-02-28

Transient evolution of light-induced molecular reorientation both in 1-amino-anthraquinone (1AAQ) dye and azobenzene doped isotropic liquid crystals (LCs) were studied by time-resolved optically heterodyned optical Kerr effect method. The results give clear direct experimental proof that under short pulse (30 ps) excitation, LC molecules orientate toward the excitation light polarization direction in the 1AAQ/LC system. However, LC molecular orientation becomes orthogonal to the light polarization in azobenzene/LC system. Time-resolved excited-state absorption of 1AAQ and wavelength dependent excited-state absorption of azobenzene were also observed and their contributions to the early dynamics of the third order optical responses of the two systems were confirmed. A simplified two-level mean-field theory was derived to reveal the intensity dependence of orientation enhancement factor in azobenzene/LC system considering the photoisomerization process.

Proteome analysis of excretory-secretory proteins of Entamoeba histolytica HM1:IMSS via LC-ESI-MS/MS and LC-MALDI-TOF/TOF.

PubMed

Ujang, Jorim Anak; Kwan, Soon Hong; Ismail, Mohd Nazri; Lim, Boon Huat; Noordin, Rahmah; Othman, Nurulhasanah

2016-01-01

Excretory-secretory (ES) proteins of E. histolytica are thought to play important roles in the host invasion, metabolism, and defence. Elucidation of the types and functions of E. histolytica ES proteins can further our understanding of the disease pathogenesis. Thus, the aim of this study is to use proteomics approach to better understand the complex ES proteins of the protozoa. E. histolytica ES proteins were prepared by culturing the trophozoites in protein-free medium. The ES proteins were identified using two mass spectrometry tools, namely, LC-ESI-MS/MS and LC-MALDI-TOF/TOF. The identified proteins were then classified according to their biological processes, molecular functions, and cellular components using the Panther classification system (PantherDB). A complementary list of 219 proteins was identified; this comprised 201 proteins detected by LC-ESI-MS/MS and 107 proteins by LC-MALDI-TOF/TOF. Of the 219 proteins, 89 were identified by both mass-spectrometry systems, while 112 and 18 proteins were detected exclusively by LC-ESI-MS/MS and LC-MALDI-TOF/TOF respectively. Biological protein functional analysis using PantherDB showed that 27% of the proteins were involved in metabolic processes. Using molecular functional and cellular component analyses, 35% of the proteins were found to be involved in catalytic activity, and 21% were associated with the cell parts. This study showed that complementary use of LC-ESI-MS/MS and LC-MALDI-TOF/TOF has improved the identification of ES proteins. The results have increased our understanding of the types of proteins excreted/secreted by the amoeba and provided further evidence of the involvement of ES proteins in intestinal colonisation and evasion of the host immune system, as well as in encystation and excystation of the parasite.

Nucleocytoplasmic Distribution and Dynamics of the Autophagosome Marker EGFP-LC3

PubMed Central

Drake, Kimberly R.; Kang, Minchul; Kenworthy, Anne K.

2010-01-01

The process of autophagy involves the formation of autophagosomes, double-membrane structures that encapsulate cytosol. Microtubule-associated protein light chain 3 (LC3) was the first protein shown to specifically label autophagosomal membranes in mammalian cells, and subsequently EGFP-LC3 has become one of the most widely utilized reporters of autophagy. Although LC3 is currently thought to function primarily in the cytosol, the site of autophagosome formation, EGFP-LC3 often appears to be enriched in the nucleoplasm relative to the cytoplasm in published fluorescence images. However, the nuclear pool of EGFP-LC3 has not been specifically studied in previous reports, and mechanisms by which LC3 shuttles between the cytoplasm and nucleoplasm are currently unknown. In this study, we therefore investigated the regulation of the nucleo-cytoplasmic distribution of EGFP-LC3 in living cells. By quantitative fluorescence microscopy analysis, we demonstrate that soluble EGFP-LC3 is indeed enriched in the nucleus relative to the cytoplasm in two commonly studied cell lines, COS-7 and HeLa. Although LC3 contains a putative nuclear export signal (NES), inhibition of active nuclear export or mutation of the NES had no effect on the nucleo-cytoplasmic distribution of EGFP-LC3. Furthermore, FRAP analysis indicates that EGFP-LC3 undergoes limited passive nucleo-cytoplasmic transport under steady state conditions, and that the diffusional mobility of EGFP-LC3 was substantially slower in the nucleus and cytoplasm than predicted for a freely diffusing monomer. Induction of autophagy led to a visible decrease in levels of soluble EGFP-LC3 relative to autophagosome-bound protein, but had only modest effects on the nucleo-cytoplasmic ratio or diffusional mobility of the remaining soluble pools of EGFP-LC3. We conclude that the enrichment of soluble EGFP-LC3 in the nucleus is maintained independently of active nuclear export or induction of autophagy. Instead, incorporation of soluble

A novel Amoeba proteus 120 kDa actin-binding protein with only 1 filamin repeat and a coiled-coil region.

PubMed

Sobczak, Magdalena; Kocik, Elzbieta; Redowicz, Maria Jolanta

2007-02-01

A novel 120 kDa actin-binding protein (ApABP-F1) was found in Amoeba proteus. It was distributed throughout the cytoplasm, mainly in the subplasma membrane and perinuclear-nuclear areas, enriched in actin. The full-length cDNA of ApABP consisted of 2672 nucleotides with an open reading frame of 878 amino acids, giving a ~95 kDa protein with a theoretical pI value of 5.11. It had a novel domain organization pattern: the N terminus (residues 1-104) contained 1 calponin-homology (CH) domain, followed by only 1 region that was homologous to the filamin repeat (FR, residues 209-324), and a central region (residues 344-577) exhibiting a very high probability of coiled-coil formation, probably engaged in the observed protein dimerization. A phylogenetic tree constructed for CH domains from 25 various proteins revealed that the CH domain of ApABP was most related to that of the hypothetical mouse KIAA0903-like protein, whereas not much relationship to either filamins or the gelation factor (ABP-120) of Dictyostelium discoideum and Entamoeba histolytica was found.

Demonstration of polarization-insensitive spatial light modulation using a single polarization-sensitive spatial light modulator.

PubMed

Liu, Jun; Wang, Jian

2015-07-06

We present a simple configuration incorporating a single polarization-sensitive phase-only liquid crystal spatial light modulator (LC-SLM) to facilitate polarization-insensitive spatial light modulation. The polarization-insensitive configuration is formed by a polarization beam splitter (PBS), a polarization-sensitive phase-only LC-SLM, a half-wave plate (HWP), and a mirror in a loop structure. We experimentally demonstrate polarization-insensitive spatial light modulations for incident linearly polarized beams with different polarization states and polarization-multiplexed beams. Polarization-insensitive spatial light modulations generating orbital angular momentum (OAM) beams are demonstrated in the experiment. The designed polarization-insensitive configuration may find promising applications in spatial light modulations accommodating diverse incident polarizations.

Biochemical characterization of the 49 kDa penicillin-binding protein of Mycobacterium smegmatis.

PubMed Central

Mukherjee, T; Basu, D; Mahapatra, S; Goffin, C; van Beeumen, J; Basu, J

1996-01-01

The 49 kDa penicillin-binding protein (PBP) of Mycobacterium smegmatis catalyses the hydrolysis of the peptide or S-ester bond of carbonyl donors R1-CONH-CHR2-COX-CHR2-COO- (where X is NH or S). In the presence of a suitable amino acceptor, the reaction partitions between the transpeptidation and hydrolysis pathways, with the amino acceptor, behaving as a simple alternative nucleophile at the level of the acyl-enzyme. By virtue of its N-terminal sequence similarity, the 49 kDa PBP represents one of the class of monofunctional low-molecular-mass PBPs. An immunologically related protein of M(r) 52,000 is present in M. tuberculosis. The 49 kDa PBP is sensitive towards amoxycillin, imipenem, flomoxef and cefoxitin. PMID:8947487

Double phosphorylation of the myosin regulatory light chain during rigor mortis of bovine Longissimus muscle.

PubMed

Muroya, Susumu; Ohnishi-Kameyama, Mayumi; Oe, Mika; Nakajima, Ikuyo; Shibata, Masahiro; Chikuni, Koichi

2007-05-16

To investigate changes in myosin light chains (MyLCs) during postmortem aging of the bovine longissimus muscle, we performed two-dimensional gel electrophoresis followed by identification with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The results of fluorescent differential gel electrophoresis showed that two spots of the myosin regulatory light chain (MyLC2) at pI values of 4.6 and 4.7 shifted toward those at pI values of 4.5 and 4.6, respectively, by 24 h postmortem when rigor mortis was completed. Meanwhile, the MyLC1 and MyLC3 spots did not change during the 14 days postmortem. Phosphoprotein-specific staining of the gels demonstrated that the MyLC2 proteins at pI values of 4.5 and 4.6 were phosphorylated. Furthermore, possible N-terminal region peptides containing one and two phosphoserine residues were detected in each mass spectrum of the MyLC2 spots at pI values of 4.5 and 4.6, respectively. These results demonstrated that MyLC2 became doubly phosphorylated during rigor formation of the bovine longissimus, suggesting involvement of the MyLC2 phosphorylation in the progress of beef rigor mortis. Bovine; myosin regulatory light chain (RLC, MyLC2); phosphorylation; rigor mortis; skeletal muscle.

The location of a disease-associated polymorphism and genomic structure of the human 52-kDa Ro/SSA locus (SSA1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsugu, H.; Horowitz, R.; Gibson, N.

1994-12-01

Sera from approximately 30% of patients with systemic lupus erythematosus (SLE) contain high titers of autoantibodies that bind to the 52-kDa Ro/SSA protein. We previously detected polymorphisms in the 52-kDa Ro/SSA gene (SSA1) with restriction enzymes, one of which is strongly associated with the presence of SLE (P < 0.0005) in African Americans. A higher disease frequency and more severe forms of the disease are commonly noted among these female patients. To determine the location and nature of this polymorphism, we obtained two clones that span 8.5 kb of the 52-kDa Ro/SSA locus including its upstream regulatory region. Six exonsmore » were identified, and their nucleotide sequences plus adjacent noncoding regions were determined. No differences were found between these exons and the coding region of one of the reported cDNAs. The disease-associated polymorphic site suggested by a restriction enzyme map and confirmed by DNA amplification and nucleotide sequencing was present upstream of exon 1. This polymorphism may be a genetic marker for a disease-related variation in the coding region for the protein or in the upstream regulatory region of this gene. Although this RFLP is present in Japanese, it is not associated with lupus in this race. 41 refs., 4 figs., 2 tabs.« less

The N-terminus and Phe52 residue of LC3 recruit p62/SQSTM1 into autophagosomes.

PubMed

Shvets, Elena; Fass, Ephraim; Scherz-Shouval, Ruthie; Elazar, Zvulun

2008-08-15

LC3 belongs to a novel ubiquitin-like protein family that is involved in different intracellular trafficking processes, including autophagy. All members of this family share a unique three-dimensional structure composed of a C-terminal ubiquitin core and two N-terminal alpha-helices. Here, we focus on the specific contribution of these regions to autophagy induced by amino acid deprivation. We show that the ubiquitin core by itself is sufficient for LC3 processing through the conjugation machinery and for its consequent targeting to the autophagosomal membrane. The N-terminal region was found to be important for interaction between LC3 and p62/SQSTM1 (hereafter termed p62). This interaction is dependent on the first 10 amino acids of LC3 and on specific residues located within the ubiquitin core. Knockdown of LC3 isoforms and overexpression of LC3 mutants that fail to interact with p62 blocked the incorporation of p62 into autophagosomes. The accumulation of p62 was accompanied by elevated levels of polyubiquitylated detergent-insoluble structures. p62, however, is not required for LC3 lipidation, autophagosome formation and targeting to lysosomes. Our results support the proposal that LC3 is responsible for recruiting p62 into autophagosomes, a process mediated by phenylalanine 52, located within the ubiquitin core, and the N-terminal region of the protein.

Molecular dissection of botulinum neurotoxin reveals interdomain chaperone function.

PubMed

Fischer, Audrey; Montal, Mauricio

2013-12-01

Clostridium botulinum neurotoxin (BoNT) is a multi-domain protein made up of the approximately 100 kDa heavy chain (HC) and the approximately 50 kDa light chain (LC). The HC can be further subdivided into two halves: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). We have investigated the minimal requirements for channel activity and LC translocation. We utilize a cellular protection assay and a single channel/single molecule LC translocation assay to characterize in real time the channel and chaperone activities of BoNT/A truncation constructs in Neuro 2A cells. The unstructured, elongated belt region of the TD is demonstrated to be dispensable for channel activity, although may be required for productive LC translocation. We show that the RBD is not necessary for channel activity or LC translocation, however it dictates the pH threshold of channel insertion into the membrane. These findings indicate that each domain functions as a chaperone for the others in addition to their individual functions, working in concert to achieve productive intoxication. Copyright © 2013 Elsevier Ltd. All rights reserved.

Therapeutic Approaches for Botulinum Intoxication Targeting Degradation of the Light Chain

DTIC Science & Technology

2013-04-01

SUBJECT TERMS Botulinum toxin , ubiquitin, chimeric toxin light chains, LcA, LcE, Yeast 2 hybrid, intracellular therapy. 16. SECURITY...Synaptic Research will develop dichain hybrids consisting of Clostridium botulinum toxin light chains (LCs) from serotypes A (long-lived) and E...stability to LCs of botulinum toxin can be assessed by mutation of dileucine residues and systematic deletion of residues from LcA-LcE chimeras to provide a

Demonstration of polarization-insensitive spatial light modulation using a single polarization-sensitive spatial light modulator

PubMed Central

Liu, Jun; Wang, Jian

2015-01-01

We present a simple configuration incorporating a single polarization-sensitive phase-only liquid crystal spatial light modulator (LC-SLM) to facilitate polarization-insensitive spatial light modulation. The polarization-insensitive configuration is formed by a polarization beam splitter (PBS), a polarization-sensitive phase-only LC-SLM, a half-wave plate (HWP), and a mirror in a loop structure. We experimentally demonstrate polarization-insensitive spatial light modulations for incident linearly polarized beams with different polarization states and polarization-multiplexed beams. Polarization-insensitive spatial light modulations generating orbital angular momentum (OAM) beams are demonstrated in the experiment. The designed polarization-insensitive configuration may find promising applications in spatial light modulations accommodating diverse incident polarizations. PMID:26146032

Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes.

PubMed

Sharma, Manish; Bhattacharyya, Sankar; Nain, Minu; Kaur, Manpreet; Sood, Vikas; Gupta, Vishal; Khasa, Renu; Abdin, Malik Z; Vrati, Sudhanshu; Kalia, Manjula

2014-09-01

Autophagy is a lysosomal degradative pathway that has diverse physiological functions and plays crucial roles in several viral infections. Here we examine the role of autophagy in the life cycle of JEV, a neurotropic flavivirus. JEV infection leads to induction of autophagy in several cell types. JEV replication was significantly enhanced in neuronal cells where autophagy was rendered dysfunctional by ATG7 depletion, and in Atg5-deficient mouse embryonic fibroblasts (MEFs), resulting in higher viral titers. Autophagy was functional during early stages of infection however it becomes dysfunctional as infection progressed resulting in accumulation of misfolded proteins. Autophagy-deficient cells were highly susceptible to virus-induced cell death. We also observed JEV replication complexes that are marked by nonstructural protein 1 (NS1) and dsRNA colocalized with endogenous LC3 but not with GFP-LC3. Colocalization of NS1 and LC3 was also observed in Atg5 deficient MEFs, which contain only the nonlipidated form of LC3. Viral replication complexes furthermore show association with a marker of the ER-associated degradation (ERAD) pathway, EDEM1 (ER degradation enhancer, mannosidase α-like 1). Our data suggest that virus replication occurs on ERAD-derived EDEM1 and LC3-I-positive structures referred to as EDEMosomes. While silencing of ERAD regulators EDEM1 and SEL1L suppressed JEV replication, LC3 depletion exerted a profound inhibition with significantly reduced RNA levels and virus titers. Our study suggests that while autophagy is primarily antiviral for JEV and might have implications for disease progression and pathogenesis of JEV, nonlipidated LC3 plays an important autophagy independent function in the virus life cycle.

[Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3].

PubMed

Wang, Wan; Zhang, Qing; Zhao, Runpeng; Xu, Xuewei; Xing, Yingru; Zhang, Rongbo; Wu, Jing; Hu, Dong

2015-09-01

To establish murine macrophage RAW264.7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3). A lentiviral vector containing RFP-GFP-LC3 gene was constructed and then packaged in HEK293T cells with the packaging plasmids. The viral supernatant was collected to infect RAW264.7 cells. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 was screened with puromycin and analyzed with flow cytometry and fluorescent microscopy for infection efficiency. The number of RFP-GFP-LC3 puncta was observed using florescence microscopy following starvation treatment. The recombinant lentivirus pLV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264.7 cells with stable expression of RFP-GFP-LC3 were obtained by viral infection and puromycin screening. Fluorescent microscopy and flow cytometry demonstrated the expression rates of RFP and GFP reached to 100%. The number of autophagic puncta significantly increased after starvation treatment. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed, which provides a reliable cellular platform for autophagy research.

Prognostic relevance of autophagy-related markers LC3, p62/sequestosome 1, Beclin-1 and ULK1 in colorectal cancer patients with respect to KRAS mutational status.

PubMed

Schmitz, Klaus Juergen; Ademi, Ceflije; Bertram, Stefanie; Schmid, Kurt Werner; Baba, Hideo Andreas

2016-07-22

Autophagy is a cellular pathway that regulates transportation of cytoplasmic macromolecules and organelles to lysosomes for degradation. Autophagy is involved in both tumorigenesis and tumour suppression. Here we investigated the potential prognostic value of the autophagy-related proteins Beclin-1, p62, LC3 and uncoordinated (UNC) 51-like kinase 1 (ULK1) in a cohort of colorectal cancer (CRC) specimens. In this study, we analysed the immunoexpression of the autophagy-related proteins p62, LC3, Beclin-1 and ULK1 in 127 CRC patients with known KRAS mutational status and detailed clinical follow-up. Survival analysis of p62 staining showed a significant correlation of cytoplasmic (not nuclear) p62 expression with a favourable tumour-specific overall survival (OS). The prognostic power of cytoplasmic p62 was found in the KRAS-mutated subgroup but was lost in the KRAS wildtype subgroup. Survival analysis of Beclin-1 staining did not show an association with OS in the complete cohort. LC3 overexpression demonstrated a slight, though not significant, association with decreased OS. Upon stratifying cases by KRAS mutational status, nuclear (not cytoplasmic) Beclin-1 staining was associated with a significantly decreased OS in the KRAS-mutated subgroup but not in the KRAS wildtype CRCs. In addition, LC3 overexpression was significantly associated with decreased OS in the KRAS-mutated CRC subgroup. ULK1 expression was not correlated to survival. Immunohistochemical analyses of LC3, p62 and Beclin-1 may constitute promising novel prognostic markers in CRC, especially in KRAS-mutated CRCs. This strategy might help in identifying high-risk patients who would benefit from autophagy-related anticancer drugs.

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism.

PubMed

Rodríguez, Andrea E; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

2016-01-01

Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism

PubMed Central

Rodríguez, Andrea E.; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

2016-01-01

ABSTRACT Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells. PMID:26654586

Promoter difference of LcFT1 is a leading cause of natural variation of flowering timing in different litchi cultivars (Litchi chinensis Sonn.).

PubMed

Ding, Feng; Zhang, Shuwei; Chen, Houbin; Su, Zuanxian; Zhang, Rong; Xiao, Qiusheng; Li, Hongli

2015-12-01

Litchi (Litchi chinensis) is an important subtropical evergreen fruit crop with high commercial value due to its high nutritional values and favorable tastes. However, irregular bearing attributed to unstable flowering is a major ongoing problem for litchi producers. There is a need to better understand the genetic and molecular mechanisms underlying the reproductive process in litchi. In a previous study, our laboratory had analyzed the transcriptome of litchi leaves before and after low-temperature treatment with RNA-seq technology. Herein, we demonstrated that litchi flowering was induced by low-temperature and identified two FLOWERING LOCUS T (FT) homologue genes named LcFT1 and LcFT2, respectively. We found that low-temperature could only induce LcFT1 expression in leaves, but could not induce LcFT2 expression. Heterologous expression of LcFT1 in transgenic tobacco and Arabidopsis plants induced their precocious flowering. These results indicate that LcFT1 plays a pivotal role in litchi floral induction by low-temperature. In addition, we found that two types of LcFT1 promoter existed in different litchi cultivars. The LcFT1 promoters in the early-flowering cultivars belonged to one type whereas LcFT1 promoters in the late-flowering belonged to another one. LcFT1 promoter in the early-flowering cultivars was more sensitive to low-temperature than that of the late-flowering cultivars was, which may be caused by the different cis-acting elements, including MYC, MYB, ABRE, and WRKY cis-acting elements, which were found to be present in the LcFT1 promoter sequences of the early-flowering cultivars. This difference may be responsible for the different requirements of low-temperature for floral induction in the early- and late-flowering cultivars of litchi. Taken together, the difference in LcFT1 promoter sequences may be one of the leading cause for the natural variation of flowering timing in different litchi cultivars. Our study has provided valuable genetic

The aqueous phase of Alzheimer's disease brain contains assemblies built from ∼4 and ∼7 kDa Aβ species.

PubMed

Mc Donald, Jessica M; O'Malley, Tiernan T; Liu, Wen; Mably, Alexandra J; Brinkmalm, Gunnar; Portelius, Erik; Wittbold, William M; Frosch, Matthew P; Walsh, Dominic M

2015-11-01

Much knowledge about amyloid β (Aβ) aggregation and toxicity has been acquired using synthetic peptides and mouse models, whereas less is known about soluble Aβ in human brain. We analyzed aqueous extracts from multiple AD brains using an array of techniques. Brains can contain at least four different Aβ assembly forms including: (i) monomers, (ii) a ∼7 kDa Aβ species, and larger species (iii) from ∼30-150 kDa, and (iv) >160 kDa. High molecular weight species are by far the most prevalent and appear to be built from ∼7 kDa Aβ species. The ∼7 kDa Aβ species resist denaturation by chaotropic agents and have a higher Aβ42/Aβ40 ratio than monomers, and are unreactive with antibodies to Asp1 of Ab or APP residues N-terminal of Asp1. Further analysis of brain-derived ∼7 kDa Aβ species, the mechanism by which they assemble and the structures they form should reveal therapeutic and diagnostic opportunities. Copyright © 2015 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

p62/SQSTM1 but not LC3 is accumulated in sarcopenic muscle of mice.

PubMed

Sakuma, Kunihiro; Kinoshita, Masakazu; Ito, Yoshinori; Aizawa, Miki; Aoi, Wataru; Yamaguchi, Akihiko

2016-05-01

We investigated the pathway of autophagy signaling linked to sarcopenia of mice. Young adult (3-month) and aged (24- month) C57BL/6J mice were used. Using real-time PCR, Western blotting, and immunohistochemical microscopy, we evaluated the amounts of p62/SQSTM1, LC3, and Beclin-1 in the quadriceps muscle change with aging in mice. Marked fiber atrophy (30%) and many fibers with central nuclei were observed in the aged mice. Western blotting using homogenate of the cytosolic fraction clearly showed that the amounts of p62/SQSTM1 and Beclin-1 proteins were significantly increased in the aged skeletal muscle. The amounts of these proteins in both nuclear and membrane fractions did not change significantly with age. Immunofluorescence labeling indicated that aged mice more frequently possessed p62/SQSTM1-positive fibers in the cytosol in quadriceps muscle than young ones (aged: 14% vs. young: 1%). In aged muscle, p62/SQSTM1-positive fibers were significantly smaller than the surrounding p62/SQSTM1-negative fibers. Aging did not elicit significant changes in the mRNA levels of p62/SQSTM1 and Beclin-1, but decreased LC3 mRNA level. In aged muscle, the location of p62/SQSTM1 immunoreactivity was similar to that of Beclin-1 protein, but not LC3. Sarcopenia in mice appears to include a marked defect of autophagy signaling.

An efficient synthesis of 1α,25-dihydroxyvitamin D3 LC-biotin.

PubMed

Kattner, Lars; Bernardi, Dan

2017-10-01

In recent years the apparent impact of vitamin D deficiency on human health has gained increased awareness. Consequently, the development of appropriate assays to measure the status of medicinally most relevant vitamin D metabolites in human blood, serum or relevant tissue is continuously being improved. Particularly, assaying of 1α,25-dihydroxyvitamin D 3 , in turn considered as the most active metabolite, is mainly indicated in disorders leading to calcaemia or those resulting from an impaired 1α-hydroxylation of 25-hydroxyvitamin D 3 . Thus, in some competitive protein binding and ELISA assays, biotin-linked 1α,25-dihydroxyvitamin D 3 (1α,25-dihydroxyvitamin D 3 LC-biotin) is employed for measurement of actual calicitriol concentration. A new efficient synthesis of 1α,25-dihydroxyvitamin D 3 LC-biotin is described, starting with readily available vitamin D 2 , and combining a classical approach to access 1α,25-dihydroxyvitamin D 3 , appropriate OH-protective group transformations, and a C-3-O-alkylation, suitable to connect the biotin-linker in a reliable, selective and high yielding strategy. The developed methodology is applicable to the synthesis of a wide variety of C-3-OH-linked vitamin D 3 and D 2 derivatives. Copyright © 2017 Elsevier Ltd. All rights reserved.

Affinity, Avidity, and Kinetics of Target Sequence Binding to LC8 Dynein Light Chain Isoforms*

PubMed Central

Radnai, László; Rapali, Péter; Hódi, Zsuzsa; Süveges, Dániel; Molnár, Tamás; Kiss, Bence; Bécsi, Bálint; Erdödi, Ferenc; Buday, László; Kardos, József; Kovács, Mihály; Nyitray, László

2010-01-01

LC8 dynein light chain (DYNLL) is a highly conserved eukaryotic hub protein with dozens of binding partners and various functions beyond being a subunit of dynein and myosin Va motor proteins. Here, we compared the kinetic and thermodynamic parameters of binding of both mammalian isoforms, DYNLL1 and DYNLL2, to two putative consensus binding motifs (KXTQTX and XG(I/V)QVD) and report only subtle differences. Peptides containing either of the above motifs bind to DYNLL2 with micromolar affinity, whereas a myosin Va peptide (lacking the conserved Gln) and the noncanonical Pak1 peptide bind with Kd values of 9 and 40 μm, respectively. Binding of the KXTQTX motif is enthalpy-driven, although that of all other peptides is both enthalpy- and entropy-driven. Moreover, the KXTQTX motif shows strikingly slower off-rate constant than the other motifs. As most DYNLL partners are homodimeric, we also assessed the binding of bivalent ligands to DYNLL2. Compared with monovalent ligands, a significant avidity effect was found as follows: Kd values of 37 and 3.5 nm for a dimeric myosin Va fragment and a Leu zipper dimerized KXTQTX motif, respectively. Ligand binding kinetics of DYNLL can best be described by a conformational selection model consisting of a slow isomerization and a rapid binding step. We also studied the binding of the phosphomimetic S88E mutant of DYNLL2 to the dimeric myosin Va fragment, and we found a significantly lower apparent Kd value (3 μm). We conclude that the thermodynamic and kinetic fine-tuning of binding of various ligands to DYNLL could have physiological relevance in its interaction network. PMID:20889982

Quantification of peptides from immunoglobulin constant and variable regions by LC-MRM MS for assessment of multiple myeloma patients.

PubMed

Remily-Wood, Elizabeth R; Benson, Kaaron; Baz, Rachid C; Chen, Y Ann; Hussein, Mohamad; Hartley-Brown, Monique A; Sprung, Robert W; Perez, Brianna; Liu, Richard Z; Yoder, Sean J; Teer, Jamie K; Eschrich, Steven A; Koomen, John M

2014-10-01

Quantitative MS assays for Igs are compared with existing clinical methods in samples from patients with plasma cell dyscrasias, for example, multiple myeloma (MM). Using LC-MS/MS data, Ig constant region peptides, and transitions were selected for LC-MRM MS. Quantitative assays were used to assess Igs in serum from 83 patients. RNA sequencing and peptide-based LC-MRM are used to define peptides for quantification of the disease-specific Ig. LC-MRM assays quantify serum levels of Igs and their isoforms (IgG1-4, IgA1-2, IgM, IgD, and IgE, as well as kappa (κ) and lambda (λ) light chains). LC-MRM quantification has been applied to single samples from a patient cohort and a longitudinal study of an IgE patient undergoing treatment, to enable comparison with existing clinical methods. Proof-of-concept data for defining and monitoring variable region peptides are provided using the H929 MM cell line and two MM patients. LC-MRM assays targeting constant region peptides determine the type and isoform of the involved Ig and quantify its expression; the LC-MRM approach has improved sensitivity compared with the current clinical method, but slightly higher inter-assay variability. Detection of variable region peptides is a promising way to improve Ig quantification, which could produce a dramatic increase in sensitivity over existing methods, and could further complement current clinical techniques. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

General Unknown Screening by Ion Trap LC/MS/MS

DTIC Science & Technology

2010-04-01

Subtitle 5 . Report Date April 2010 General Unknown Screening by Ion Trap LC/MS/MS 6 . Performing Organization Code 7. Author(s) 8... 5 Table 1: Analytical Data for Each of the...359 Compounds in the LC/MS/MS Library . . . . . . . . . . . 6 1 General Unknown ScreeninG by ion Trap lc/MS/MS INTrOduCTION The Federal Aviation

Clinicopathological Profiling of LC3B, an Autophagy Marker, and ESRRA (Estrogen-related Receptor-alpha) in Muscle-invasive Bladder Cancer.

PubMed

Kim, Sup; Lee, Adam Jaehyeok; Yeo, Min-Kyung; Na, Yong Gil; Kim, Ji-Yeon; Cho, Moon-June; Kim, Jun-Sang; Jo, Eun-Kyeong; Kim, Jin-Man

2018-04-01

Microtubule-associated protein 1 light chain 3B (LC3B), an autophagy marker, has been used as a promising marker in various cancer types. However, the expression of LC3B in muscle-invasive bladder cancer (MIBC) and its prognostic significance have not been investigated. Recent studies pointed to the involvement of ESRRA in regulating autophagy via both transcriptional and post-translational control. In the current study, prognostic importance of LC3B and ESRRA in MIBC was investigated. We immunohistochemically studied the expression of LC3B and ESRRA in 56 MIBC samples. LC3B was stained high in 16 patients (28.6%) and low or negative in 40 patients (71.4%). ESRRA expression was high for 20 patients (35.7%) and low for 36 patients (64.3%). Both LC3B (p=0.003) and ESRRA (p=0.026) expression correlated significantly with disease-free survival rates. Double-positive LC3B and ESRRA correlated with poor overall survival (p=0.007) and disease-free survival (p=0.001) in MIBC patients. LC3B and ESRRA might be a useful prognostic factor in patients with MIBC. The co-expression of LC3B and ESRRA might be a prognostic and therapeutic target for patients with bladder cancer. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein.

PubMed

Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

2016-03-01

Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome

Effects of porcine 25 kDa amelogenin and its proteolytic derivatives on bone sialoprotein expression.

PubMed

Nakayama, Y; Yang, L; Mezawa, M; Araki, S; Li, Z; Wang, Z; Sasaki, Y; Takai, H; Nakao, S; Fukae, M; Ogata, Y

2010-10-01

Amelogenins are hydrophobic proteins that are the major component of developing enamel. Enamel matrix derivative has been used for periodontal regeneration. Bone sialoprotein is an early phenotypic marker of osteoblast differentiation. In this study, we examined the ability of porcine amelogenins to regulate bone sialoprotein transcription. To determine the molecular basis of the transcriptional regulation of the bone sialoprotein gene by amelogenins, we conducted northern hybridization, transient transfection analyses and gel mobility shift assays using the osteoblast-like ROS 17/2.8 cells. Amelogenins (100 ng/mL) up-regulated bone sialoprotein mRNA at 3 h, with maximal mRNA expression occurring at 12 h (25 and 20 kDa) and 6 h (13 and 6 kDa). Amelogenins (100 ng/mL, 12 h) increased luciferase activities in pLUC3 (nucleotides -116 to +60), and 6 kDa amelogenin up-regulated pLUC4 (nucleotides -425 to +60) activity. The tyrosine kinase inhibitor inhibited amelogenin-induced luciferase activities, whereas the protein kinase A inhibitor abolished 25 kDa amelogenin-induced bone sialoprotein transcription. The effects of amelogenins were abrogated by 2-bp mutations in the fibroblast growth factor 2 response element (FRE). Gel-shift assays with radiolabeled FRE, homeodomain-protein binding site (HOX) and transforming growth factor-beta1 activation element (TAE) double-strand oligonucleotides revealed increased binding of nuclear proteins from amelogenin-stimulated ROS 17/2.8 cells at 3 h (25 and 13 kDa) and 6 h (20 and 6 kDa). These results demonstrate that porcine 25 kDa amelogenin and its proteolytic derivatives stimulate bone sialoprotein transcription by targeting FRE, HOX and TAE in the bone sialoprotein gene promoter, and that full-length amelogenin and amelogenin cleavage products are able to regulate bone sialoprotein transcription via different signaling pathways. (c) 2010 John Wiley & Sons A/S.

Autophagy-independent incorporation of GFP-LC3 into protein aggregates is dependent on its interaction with p62/SQSTM1.

PubMed

Shvets, Elena; Elazar, Zvulun

2008-11-01

LC3 is a widely used marker of autophagosomes in mammalian cells. However, in addition to its autophagosomal localization, GFP-LC3 is often found associated with protein aggregates that are formed in an autophagy-independent manner. In addition, LC3 directly interacts with p62/SQSTM1 (hereafter named p62), a common constituent of protein aggregates. In our recent report, we mapped the regions in LC3 involved in its binding to p62 and showed that this binding is essential for the incorporation of p62 into autophagosomes. Here we demonstrate that the autophagy-unrelated association of GFP-LC3 with protein aggregates is dependent on its interaction with p62.

Trichinella spiralis: strong antibody response to a 49 kDa newborn larva antigen in infected rats.

PubMed

Salinas-Tobon, Maria Del Rosario; Navarrete-Leon, Anaid; Mendez-Loredo, Blanca Esther; Esquivel-Aguirre, Dalia; Martínez-Abrajan, Dulce Maria; Hernandez-Sanchez, Javier

2007-02-01

In this work, we analyzed the kinetics of anti-Trichinella spiralis newborn larva (NBL) antibodies (Ab) and the antigenic recognition pattern of NBL proteins and its dose effects. Wistar rats were infected with 0, 700, 2000, 4000 and 8000 muscle larvae (ML) and bled at different time intervals up to day 31 post infection (p.i.). Ab production was higher with 2000 ML dose and decreased with 8000, 4000 and 700 ML. Abs were not detected until day 10, peaked on day 14 for the 2000 ML dose and on day 19 for the other doses and thereafter declined slowly from 19 to 31 days p.i. In contrast, Abs to ML increased from day 10, peaked on day 19 and remained high until the end of the study. Abs bound strongly at least to three NBL components of 188, 205 and 49 kDa. NBL antigen of 188 and 205 kDa were recognized 10-26 days p.i. and that of 49 kDa from day 10 to day 31 p.i. A weak recognition towards antigens of 52, 54, 62 and 83 kDa was also observed during the infection. An early recognition of 31, 43, 45, 55, 68 and 85 kDa ML antigens was observed whereas the response to those of 43, 45, 48, 60, 64 and 97 kDa (described previously as TSL-1 antigens) occurred late in the infection. A follow-up of antigen recognition up to day 61 with the optimal immunization dose (2000 ML) evidenced a decline of Ab production to the 49 kDa NBL antigen 42 days p.i., which suggested antigenic differences with the previously reported 43 kDa ML antigen strongly recognized late in the infection. To analyze the stage-specificity of the 49 kDa NBL antigen, polyclonal antibodies (PoAb) were obtained in rats immunized with 49 kDa NBL antigen. PoAb reacted strongly with the 49 kDa NBL component in NBL total soluble extract but no reactivity was observed with soluble antigen of the other T. spiralis stages. Albeit with less intensity, the 49 kDa component was also recognized by PoAb together with other antigens of 53, 97 and 107 kDa, in NBL excretory-secretory products (NBL-ESP). Thus, our results reveal

Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting.

PubMed

Zeman, David; Kušnierová, Pavlína; Švagera, Zdeněk; Všianský, František; Byrtusová, Monika; Hradílek, Pavel; Kurková, Barbora; Zapletalová, Olga; Bartoš, Vladimír

2016-01-01

We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.

Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting

PubMed Central

Kušnierová, Pavlína; Švagera, Zdeněk; Všianský, František; Byrtusová, Monika; Hradílek, Pavel; Kurková, Barbora; Zapletalová, Olga; Bartoš, Vladimír

2016-01-01

Objectives We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. Methods We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. Results Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. Conclusions Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance. PMID:27846293

FmLC5, a putative galactose-binding C-type lectin with two QPD motifs from the hemocytes of Fenneropenaeus merguiensis participates in shrimp immune defense.

PubMed

Senghoi, Wilaiwan; Runsaeng, Phanthipha; Utarabhand, Prapaporn

2017-11-01

Crustaceans are deficient in adaptive immune system. They depend completely on an innate immunity to protect themselves from invading microorganisms. One kind of pattern recognition receptors that contribute roles in the innate immunity is lectin. A new C-type lectin gene designated as FmLC5 was isolated from Fenneropenaeus merguiensis. Its full-length cDNA is composed of 1526bp and one open reading frame of 852bp encoding a peptide of 284 amino acids. The deduced amino acid sequence of FmLC5 comprises a signal peptide of 20 contiguous amino acids with a molecular mass of 31.47kDa and an isoelectric point of 4.35. The primary structure of FmLC5 consists of two similar carbohydrate recognition domains (CRDs), each CRD contains a Ca 2+ binding site-2 and a QPD motif specific for galactose-binding. The FmLC5 transcripts were detected only in the hemocytes analyzed by RT-PCR and in situ hybridization. The FmLC5 expression was significantly up-regulated after challenge with Vibrio harveyi, white spot syndrome virus (WSSV) or lipopolysaccharide. RNAi-based silencing with co-injection by V. harveyi or WSSV resulted in critical suppression of the FmLC5 expression, increasing in mortality and reduction of the median lethal time. These results conclude that FmLC5 is unique putative galactose-binding C-type lectin in F. merguiensis that may contribute as receptor molecule in the immune response to defend the shrimp from pathogenic bacteria and viruses. Copyright © 2017 Elsevier Inc. All rights reserved.

8-Hydroxyquinoline and hydroxamic acid inhibitors of botulinum neurotoxin BoNT/A.

PubMed

Dickerson, Tobin J; Smith, Garry R; Pelletier, Jeffrey C; Reitz, Allen B

2014-01-01

We describe here the state of the art of certain aspects concerning potential small molecule therapy directed toward botulism, by inhibition of the zinc-protease containing light chain (LC) of botulinum neurotoxin BoNT/A from the anaerobic bacillus Clostridium botulinum. Botulinum neurotoxins (BoNTs) are comprised of eight serologically-distinct proteins (A - H), several of which are further divided, such as BoNT/A which has five subtypes. The BoNTs are the most toxic substances known to mankind, causing a form of flaccid paralysis that can be rapid and is often lethal. BoNT/A is comprised of a ~100 kDa heavy chain (HC) attached via a single disulfide Cys-Cys bond to a ~50 kDa LC. The HC mediates transport to and uptake by presynaptic glutamatergic neurons, where the LC cleaves the protein SNAP-25 and thus prevents vesicular trafficking and release of acetylcholine. The Zn-endoprotease activity of the LC of BoNT/A is a target for the development of small molecule inhibitors of BoNT/A-mediated toxicity. A variety of BoNT/A LC inhibitors have been described to date and we focus here primarily on the Zn-binding 8-hydroxyquinoline structural type as well as some of the previously-described hydroxamic acids.

Molecular composition and extinction coefficient of native botulinum neurotoxin complex produced by Clostridium botulinum hall A strain.

PubMed

Bryant, Anne-Marie; Davis, Jenny; Cai, Shuowei; Singh, Bal Ram

2013-02-01

Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)(-1)cm(-1). These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.

Carboxyl methylation of 21-23 kDa membrane proteins in intact neuroblastoma cells is increased with differentiation.

PubMed

Haklai, R; Kloog, Y

1990-01-01

Evidence is presented for specific enzymatic methylation of 21-23 kDa membrane proteins in intact neuroblastoma N1E 115 cells, which is increased in dimethylsulfoxide-induced differentiated cells. Methylation of these proteins has characteristics typical of enzymatic reactions in which base labile volatile methyl groups are incorporated into proteins, consistent with the formation of protein carboxyl methylesters. However, these methylesters of the 21-23 kDa proteins are relatively stable compared to other protein carboxyl methylesters. The 3-fold increase in methylated 21-23 kDa proteins in the differentiated cells suggest biological significance in differentiation of the cell membranes.

User Guide to the 1981 LC (Language Census) Database. Version 1.2.

ERIC Educational Resources Information Center

Kimbrough, Kenneth L.

This guide is designed to introduce potential users of the California Language Census data to a means of accessing that data using an online, interactive computer system known as "1981 LC." The language census is an actual count of the numbers of pupils with a primary language other than English in California public schools as of March 1…

Biofortification of soybean meal: immunological properties of the 27 kDa γ-zein.

PubMed

Krishnan, Hari B; Jang, Sungchan; Kim, Won-Seok; Kerley, Monty S; Oliver, Melvin J; Trick, Harold N

2011-02-23

Legumes, including soybeans ( Glycine max ), are deficient in sulfur-containing amino acids, which are required for the optimal growth of monogastric animals. This deficiency can be overcome by expressing heterologous proteins rich in sulfur-containing amino acids in soybean seeds. A maize 27 kDa γ-zein, a cysteine-rich protein, has been successfully expressed in several crops including soybean, barley, and alfalfa with the intent to biofortify these crops for animal feed. Previous work has shown that the maize 27 kDa zein can withstand digestion by pepsin and elicit an immunogenic response in young pigs. By use of sera from patients who tested positive by ImmunoCAP assay for elevated IgE to maize proteins, specific IgE binding to the 27 kDa γ-zein is demonstrated. Bioinformatic analysis using the full-length and 80 amino acid sliding window FASTA searches identified significant sequence homology of the 27 kDa γ-zein with several known allergens. Immunoblot analysis using human serum that cross-reacts with maize seed proteins also revealed specific IgE-binding to the 27 kDa γ-zein in soybean seed protein extracts containing the 27 kDa zein. This study demonstrates for the first time the allergenicity potential of the 27 kDa γ-zein and the potential that this protein has to limit livestock performance when used in soybeans that serve as a biofortified feed supplement.

A Bayesian framework based on a Gaussian mixture model and radial-basis-function Fisher discriminant analysis (BayGmmKda V1.1) for spatial prediction of floods

NASA Astrophysics Data System (ADS)

Tien Bui, Dieu; Hoang, Nhat-Duc

2017-09-01

In this study, a probabilistic model, named as BayGmmKda, is proposed for flood susceptibility assessment in a study area in central Vietnam. The new model is a Bayesian framework constructed by a combination of a Gaussian mixture model (GMM), radial-basis-function Fisher discriminant analysis (RBFDA), and a geographic information system (GIS) database. In the Bayesian framework, GMM is used for modeling the data distribution of flood-influencing factors in the GIS database, whereas RBFDA is utilized to construct a latent variable that aims at enhancing the model performance. As a result, the posterior probabilistic output of the BayGmmKda model is used as flood susceptibility index. Experiment results showed that the proposed hybrid framework is superior to other benchmark models, including the adaptive neuro-fuzzy inference system and the support vector machine. To facilitate the model implementation, a software program of BayGmmKda has been developed in MATLAB. The BayGmmKda program can accurately establish a flood susceptibility map for the study region. Accordingly, local authorities can overlay this susceptibility map onto various land-use maps for the purpose of land-use planning or management.

Inverse correlation between microtubule-associated protein 1A/1B-light chain 3 and p62/sequestosome-1 expression in the progression of cutaneous squamous cell carcinoma.

PubMed

Yoshihara, Nagisa; Takagi, Atsushi; Ueno, Takashi; Ikeda, Shigaku

2014-04-01

The expression of autophagy-related markers has occasionally been reported to correlate with the clinical stage of disease in patients with solid cancer, indicating autophagy activation. However, there have been no such reports for cutaneous squamous cell carcinoma. In this study, we investigated the expression levels of two autophagy-related markers, microtubule-associated protein IA/IB light chain 3 (LC3) and p62/sequestosome-1 (p62), in cutaneous squamous cell carcinoma specimens and assessed their correlation to clinicopathological factors in patients with this type of cancer. As a marker of the autophagosome, LC3 expression increases with autophagosome formation/accumulation, whereas p62 expression decreases due to selective degradation via autophagy. We performed immunostaining for LC3 and p62 in 50 cutaneous squamous cell carcinoma specimens obtained from patients treated by surgical resection, counted the number of cells that showed positive staining, and calculated the percentage of positive cells per low-power microscopic field. We next investigated the correlations between the expression levels of these markers and various clinicopathological factors. The results indicated that LC3 expression increased significantly with advanced clinical stage (P < 0.001) and increased tumor diameter (P = 0.046). By contrast, the expression of p62 decreased significantly with advanced clinical stage (P < 0.001) and increased tumor diameter (P = 0.001). These results suggest that autophagy becomes activated during disease progression in patients with cutaneous squamous cell carcinoma. © 2014 Japanese Dermatological Association.

Sunitinib induces genomic instability of renal carcinoma cells through affecting the interaction of LC3-II and PARP-1.

PubMed

Yan, Siyuan; Liu, Ling; Ren, Fengxia; Gao, Quan; Xu, Shanshan; Hou, Bolin; Wang, Yange; Jiang, Xuejun; Che, Yongsheng

2017-08-10

Deficiency of autophagy has been linked to increase in nuclear instability, but the role of autophagy in regulating the formation and elimination of micronuclei, a diagnostic marker for genomic instability, is limited in mammalian cells. Utilizing immunostaining and subcellular fractionation, we found that either LC3-II or the phosphorylated Ulk1 localized in nuclei, and immunoprecipitation results showed that both LC3 and Unc-51-like kinase 1 (Ulk1) interacted with γ-H2AX, a marker for the DNA double-strand breaks (DSB). Sunitinib, a multi-targeted receptor tyrosine kinase inhibitor, was found to enhance the autophagic flux concurring with increase in the frequency of micronuclei accrued upon inhibition of autophagy, and similar results were also obtained in the rasfonin-treated cells. Moreover, the punctate LC3 staining colocalized with micronuclei. Unexpectedly, deprivation of SQSTM1/p62 alone accumulated micronuclei, which was not further increased upon challenge with ST. Rad51 is a protein central to repairing DSB by homologous recombination and treatment with ST or rasfonin decreased its expression. In several cell lines, p62 appeared in the immunoprecipites of Rad51, whereas LC3, Ulk1 and p62 interacted with PARP-1, another protein involved in DNA repair and genomic stability. In addition, knockdown of either Rad51 or PARP-1 completely inhibited the ST-induced autophagic flux. Taken together, the data presented here demonstrated that both LC3-II and the phosphorylated Ulk1 localized in nuclei and interacted with the proteins essential for nuclear stability, thereby revealing a more intimate relationship between autophagy and genomic stability.

Sunitinib induces genomic instability of renal carcinoma cells through affecting the interaction of LC3-II and PARP-1

PubMed Central

Yan, Siyuan; Liu, Ling; Ren, Fengxia; Gao, Quan; Xu, Shanshan; Hou, Bolin; Wang, Yange; Jiang, Xuejun; Che, Yongsheng

2017-01-01

Deficiency of autophagy has been linked to increase in nuclear instability, but the role of autophagy in regulating the formation and elimination of micronuclei, a diagnostic marker for genomic instability, is limited in mammalian cells. Utilizing immunostaining and subcellular fractionation, we found that either LC3-II or the phosphorylated Ulk1 localized in nuclei, and immunoprecipitation results showed that both LC3 and Unc-51-like kinase 1 (Ulk1) interacted with γ-H2AX, a marker for the DNA double-strand breaks (DSB). Sunitinib, a multi-targeted receptor tyrosine kinase inhibitor, was found to enhance the autophagic flux concurring with increase in the frequency of micronuclei accrued upon inhibition of autophagy, and similar results were also obtained in the rasfonin-treated cells. Moreover, the punctate LC3 staining colocalized with micronuclei. Unexpectedly, deprivation of SQSTM1/p62 alone accumulated micronuclei, which was not further increased upon challenge with ST. Rad51 is a protein central to repairing DSB by homologous recombination and treatment with ST or rasfonin decreased its expression. In several cell lines, p62 appeared in the immunoprecipites of Rad51, whereas LC3, Ulk1 and p62 interacted with PARP-1, another protein involved in DNA repair and genomic stability. In addition, knockdown of either Rad51 or PARP-1 completely inhibited the ST-induced autophagic flux. Taken together, the data presented here demonstrated that both LC3-II and the phosphorylated Ulk1 localized in nuclei and interacted with the proteins essential for nuclear stability, thereby revealing a more intimate relationship between autophagy and genomic stability. PMID:28796254

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

PubMed Central

Radford, N B; Fina, M; Benjamin, I J; Moreadith, R W; Graves, K H; Zhao, P; Gavva, S; Wiethoff, A; Sherry, A D; Malloy, C R; Williams, R S

1996-01-01

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart. Images Fig. 1 Fig. 3 PMID:8637874

22. INTERIOR OR BEDROOM NO. 1 SHOWING 1LIGHT OVER 1LIGHT, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

22. INTERIOR OR BEDROOM NO. 1 SHOWING 1-LIGHT OVER 1-LIGHT, DOUBLE-HUNG WINDOW ON EAST WALL. VIEW TO SOUTHEAST. - Bishop Creek Hydroelectric System, Plant 6, Cashbaugh-Kilpatrick House, Bishop Creek, Bishop, Inyo County, CA

Accurate LC Peak Boundary Detection for 16 O/ 18 O Labeled LC-MS Data

PubMed Central

Cui, Jian; Petritis, Konstantinos; Tegeler, Tony; Petritis, Brianne; Ma, Xuepo; Jin, Yufang; Gao, Shou-Jiang (SJ); Zhang, Jianqiu (Michelle)

2013-01-01

In liquid chromatography-mass spectrometry (LC-MS), parts of LC peaks are often corrupted by their co-eluting peptides, which results in increased quantification variance. In this paper, we propose to apply accurate LC peak boundary detection to remove the corrupted part of LC peaks. Accurate LC peak boundary detection is achieved by checking the consistency of intensity patterns within peptide elution time ranges. In addition, we remove peptides with erroneous mass assignment through model fitness check, which compares observed intensity patterns to theoretically constructed ones. The proposed algorithm can significantly improve the accuracy and precision of peptide ratio measurements. PMID:24115998

Directed modification of L-LcLDH1, an L-lactate dehydrogenase from Lactobacillus casei, to improve its specific activity and catalytic efficiency towards phenylpyruvic acid.

PubMed

Li, Jian-Fang; Li, Xue-Qing; Liu, Yan; Yuan, Feng-Jiao; Zhang, Ting; Wu, Min-Chen; Zhang, Ji-Ru

2018-05-22

To improve the specific activity and catalytic efficiency of L-LcLDH1, an NADH-dependent allosteric L-lactate dehydrogenase from L. casei, towards phenylpyruvic acid (PPA), its directed modification was conducted based on the semi-rational design. The three variant genes, Lcldh1 Q88R , Lcldh1 I229A and Lcldh1 T235G , were constructed by whole-plasmid PCR as designed theoretically, and expressed in E. coli BL21(DE3), respectively. The purified mutant, L-LcLDH1 Q88R or L-LcLDH1 I229A , displayed the specific activity of 451.5 or 512.4 U/mg towards PPA, by which the asymmetric reduction of PPA afforded L-phenyllactic acid (PLA) with an enantiomeric excess (ee p ) more than 99%. Their catalytic efficiencies (k cat /K m ) without D-fructose-1,6-diphosphate (D-FDP) were 4.8- and 5.2-fold that of L-LcLDH1. Additionally, the k cat /K m values of L-LcLDH1 Q88R and L-LcLDH1 I229A with D-FDP were 168.4- and 8.5-fold higher than those of the same enzymes without D-FDP, respectively. The analysis of catalytic mechanisms by molecular docking (MD) simulation indicated that substituting I229 in L-LcLDH1 with Ala enlarges the space of substrate-binding pocket, and that the replacement of Q88 with Arg makes the inlet of pocket larger than that of L-LcLDH1. Copyright © 2018 Elsevier B.V. All rights reserved.

Human microvascular dysfunction and apoptotic injury induced by AL amyloidosis light chain proteins.

PubMed

Migrino, Raymond Q; Truran, Seth; Gutterman, David D; Franco, Daniel A; Bright, Megan; Schlundt, Brittany; Timmons, Mitchell; Motta, Angelica; Phillips, Shane A; Hari, Parameswaran

2011-12-01

Light chain amyloidosis (AL) involves overproduction of amyloidogenic light chain proteins (LC) leading to heart failure, yet the mechanisms underlying tissue toxicity remain unknown. We hypothesized that LC induces endothelial dysfunction in non-AL human microvasculature and apoptotic injury in human coronary artery endothelial cells (HCAECs). Adipose arterioles (n = 34, 50 ± 3 yr) and atrial coronary arterioles (n = 19, 68 ± 2 yr) from non-AL subjects were cannulated. Adipose arteriole dilator responses to acetylcholine/papaverine were measured at baseline and 1 h exposure to LC (20 μg/ml) from biopsy-proven AL subjects (57 ± 11 yr) without and with antioxidant cotreatment. Coronary arteriole dilation to bradykinin/papaverine was measured post-LC exposure. HCAECs were exposed to 1 or 24 h of LC. LC reduced dilation to acetylcholine (10(-4) M: 41.6 ± 7 vs. 85.8 ± 2.2% control, P < 0.001) and papaverine (81.4 ± 4.6 vs. 94.8 ± 1.3% control, P < 0.01) in adipose arterioles and to bradykinin (10(-6) M: 68.6 ± 6.2 vs. 90.9 ± 1.6% control, P < 0.001) but not papaverine in coronary arterioles. There was an increase in superoxide and peroxynitrite in arterioles treated with LC. Adipose arteriole dilation was restored by cotreatment with polyethylene glycol-superoxide dismutase and tetrahydrobiopterin but only partially restored by mitoquinone (mitochondria-targeted antioxidant) and gp91ds-tat (NADPH oxidase inhibitor). HCAECs exposed to LC showed reduced NO and increased superoxide, peroxynitrite, annexin-V, and propidium iodide compared with control. Brief exposure to physiological amounts of LC induced endothelial dysfunction in human adipose and coronary arterioles and increased apoptotic injury in coronary artery endothelial cells likely as a result of oxidative stress, reduced NO bioavailability, and peroxynitrite production. Microvascular dysfunction and injury is a novel mechanism underlying AL pathobiology and is a potential target for therapy.

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry.

PubMed

Pugsley, Haley R

2017-07-21

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

PubMed Central

Pugsley, Haley R.

2017-01-01

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner. PMID:28784946

Cloning and Characterization of Lxr and Srebp1, and Their Potential Roles in Regulation of LC-PUFA Biosynthesis in Rabbitfish Siganus canaliculatus.

PubMed

Zhang, Qinghao; You, Cuihong; Liu, Fang; Zhu, Wendi; Wang, Shuqi; Xie, Dizhi; Monroig, Óscar; Tocher, Douglas R; Li, Yuanyou

2016-09-01

Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability to biosynthesize C20-22 long-chain polyunsaturated fatty acid (LC-PUFA) from C18 PUFA precursors, which is generally absent or low in marine teleosts. Thus, understanding the molecular basis of LC-PUFA biosynthesis in rabbitfish will contribute to efforts aimed at optimizing LC-PUFA biosynthesis in teleosts, especially marine species. In the present study, the importance of the transcription factors liver X receptor (Lxr) and sterol regulatory element-binding protein 1 (Srebp1) in regulation of LC-PUFA biosynthesis in rabbitfish was investigated. First, full-length cDNA of Lxr and Srebp1 were cloned and characterized. The Lxr mRNA displayed a ubiquitous tissue expression pattern while Srebp1 was highly expressed in eyes, brain and intestine. In rabbitfish primary hepatocytes treated with Lxr agonist T0901317, the expression of Lxr and Srebp1 was activated, accompanied by elevated mRNA levels of Δ4 and Δ6/Δ5 fatty acyl desaturase (Fad), key enzymes of LC-PUFA biosynthesis, as well as peroxisome proliferator-activated receptor γ (PPARγ). In addition, Srebp1 displayed higher expression levels in liver of rabbitfish fed a vegetable oil diet or reared at 10 ppt salinity, which were conditions reported to increase the liver expression of Δ4 and Δ6/Δ5 Fad and LC-PUFA biosynthetic ability, than fish fed a fish oil diet or reared at 32 ppt, respectively. These results suggested that Lxr and Srebp1 are involved in regulation of LC-PUFA biosynthesis probably by promoting the expression of two Fad in rabbitfish liver, which, to our knowledge, is the first report in marine teleosts.

Influence of 120 kDa Pyruvate:Ferredoxin Oxidoreductase on Pathogenicity of Trichomonas vaginalis.

PubMed

Song, Hyun-Ouk

2016-02-01

Trichomonas vaginalis is a flagellate protozoan parasite and commonly infected the lower genital tract in women and men. Iron is a known nutrient for growth of various pathogens, and also reported to be involved in establishment of trichomoniasis. However, the exact mechanism was not clarified. In this study, the author investigated whether the 120 kDa protein of T. vaginalis may be involved in pathogenicity of trichomonads. Antibodies against 120 kDa protein of T. vaginalis, which was identified as pyruvate:ferredoxin oxidoreductase (PFOR) by peptide analysis of MALDI-TOF-MS, were prepared in rabbits. Pretreatment of T. vaginalis with anti-120 kDa Ab decreased the proliferation and adherence to vaginal epithelial cells (MS74) of T. vaginalis. Subcutaneous tissue abscess in anti-120 kDa Ab-treated T. vaginalis-injected mice was smaller in size than that of untreated T. vaginalis-infected mice. Collectively, the 120 kDa protein expressed by iron may be involved in proliferation, adhesion to host cells, and abscess formation, thereby may influence on the pathogenicity of T. vaginalis.

Studying the highly bent spectra of FR II-type radio galaxies with the KDA EXT model

NASA Astrophysics Data System (ADS)

Kuligowska, Elżbieta

2018-04-01

Context. The Kaiser, Dennett-Thorpe & Alexander (KDA, 1997, MNRAS, 292, 723) EXT model, that is, the extension of the KDA model of Fanaroff & Riley (FR) II-type source evolution, is applied and confronted with the observational data for selected FR II-type radio sources with significantly aged radio spectra. Aim. A sample of FR II-type radio galaxies with radio spectra strongly bent at their highest frequencies is used for testing the usefulness of the KDA EXT model. Methods: The dynamical evolution of FR II-type sources predicted with the KDA EXT model is briefly presented and discussed. The results are then compared to the ones obtained with the classical KDA approach, assuming the source's continuous injection and self-similarity. Results: The results and corresponding diagrams obtained for the eight sample sources indicate that the KDA EXT model predicts the observed radio spectra significantly better than the best spectral fit provided by the original KDA model.

Th1-stimulatory polyproteins of soluble Leishmania donovani promastigotes ranging from 89.9 to 97.1 kDa offers long-lasting protection against experimental visceral leishmaniasis.

PubMed

Kumari, Shraddha; Samant, Mukesh; Misra, Pragya; Khare, Prashant; Sisodia, Brijesh; Shasany, Ajit K; Dube, Anuradha

2008-10-23

Our earlier studies identified a fraction (F2) of Leishmania donovani soluble promastigote antigen belonging to 97.4-68 kDa for its ability to stimulate Th1-type cellular responses in cured visceral leishmaniasis (VL) patients as well as in cured hamsters. A further fractionation of F2-fraction into seven subfractions (F2.1-F2.7) and re-assessment for their immunostimulatory responses revealed that out of these, only four (F2.4-F2.7) belonging to 89.9-97.1 kDa, stimulated remarkable Th1-type cellular responses either individually or in a pooled form (P4-7). In this study these potential subfractions were further assessed for their prophylactic potential in combination with BCG against L. donovani challenge in hamsters. Optimum parasite inhibition ( approximately 99%) was obtained in hamsters vaccinated with pooled subfractions and they survived for 1 year. The protection was further supported by remarkable lymphoproliferative, IFN-gamma and IL-12 responses along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody as observed on days 45, 90 and 120 post-challenge suggesting that a successful subunit vaccine against VL may require multiple Th1-immunostimulatory proteins. MALDI-TOF-MS/MS analysis of these subfractions further revealed that of the 19 identified immunostimulatory proteins, Elongation factor-2, p45, Heat shock protein-70/83, Aldolase, Enolase, Triosephosphate isomerase, Disulfideisomerase and Calreticulin were the major ones in these subfractions.

Polymerization speed and diffractive experiments in polymer network LC test cells

NASA Astrophysics Data System (ADS)

Braun, Larissa; Gong, Zhen; Habibpourmoghadam, Atefeh; Schafforz, Samuel L.; Wolfram, Lukas; Lorenz, Alexander

2018-02-01

Polymer-network liquid crystals (LCs), where the response properties of a LC can be enhanced by the presence of a porous polymer network, are investigated. In the reported experiments, liquid crystals were doped with a small amount (< 10%) of photo-curable acrylate monomers. Samples with surface grafted photoinitiators, dissolvable photoinitiators, and samples with both kinds of photoinitiators were prepared. Both conventional (planar electrodes) and diffractive (interdigitated electrodes) test cells were used. These samples were exposed with a UV light source and changes of their capacitance were investigated with an LCR meter during exposure. Due to the presence of the in-situ generated polymer network, the electro-optic response properties of photo cured samples were enhanced. For example, their continuous phase modulation properties led to more localized responses in samples with interdigitated electrodes, which caused suppression of selected diffraction orders in the diffraction patterns recorded in polymer network LC samples. Moreover, capacitance changes were investigated during photopolymerization of a blue phase LC.

Coupling of Ultrafast LC with Mass Spectrometry by DESI

NASA Astrophysics Data System (ADS)

Cai, Yi; Liu, Yong; Helmy, Roy; Chen, Hao

2014-10-01

Recently we reported a desorption electrospray ionization (DESI) interface to combine liquid chromatography (LC) with mass spectrometry (MS) using a new LC eluent splitting strategy through a tiny orifice on LC capillary tube [ J. Am. Soc. Mass Spectrom. 25, 286 (2014)]. The interface introduces negligible dead volume and back pressure, thereby allowing "near real-time" MS detection, fast LC elution, and online MS-directed purification. This study further evaluates the LC/DESI-MS performance with focus of using ultra-fast LC. Using a monolithic C18 column, metabolites in urine can be separated within 1.6 min and can be online collected for subsequent structure elucidation (e.g., by NMR, UV, IR) in a recovery yield up to 99%. Using a spray solvent with alkaline pH, negative ions could be directly generated for acidic analytes (e.g., ibuprofen) in acidic LC eluent by DESI, offering a novel protocol to realize "wrong-way around" ionization for LC/MS analysis. In addition, DESI-MS is found to be compatible with ultra-performance liquid chromatography (UPLC) for the first time.

Expression of autophagy-related protein LC3B, p62, and cytoplasmic p53 in human retinoblastoma tissues.

PubMed

Zhang, M; Zhou, Y-F; Gong, J-Y; Gao, C-B; Li, S-L

2016-07-01

Dysfunction of autophagy has been implicated in development and progression of diverse human cancers. However, the exact role and mechanism of autophagy have not been fully understood in human cancers, especially in retinoblastoma (Rb). We determined the autophagy activity in human Rb tissues by assessing the autophagy markers microtubule-associated protein light chain 3B (LC3) and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry and then associated their expression with patient clinicopathological features. We further explored the correlation between the expression of LC3B and p62 and the expression of cytoplasmic p53, a newly identified autophagy suppressor, in Rb tissues. Our data revealed that the expression of LC3B and p62, was significantly associated with disease progression and tumor invasion of Rb. Furthermore, we also revealed that cytoplasmic expression of p53 was inversely associated with the behavior of tumor invasion. Finally, Spearman correlation analysis demonstrated that cytoplasmic expression of p53 was significantly and inversely correlated to the expression of both LC3B and p62. Autophagy might play an important role in human Rb progression, and LC3B and p62 may be useful predictors of disease progression in patients with Rb.

Spatial resolution limitation of liquid crystal spatial light modulator

NASA Astrophysics Data System (ADS)

Wang, Xinghua; Wang, Bin; McManamon, Paul F., III; Pouch, John J.; Miranda, Felix A.; Anderson, James E.; Bos, Philip J.

2004-10-01

The effect of fringing electric fields in a liquid crystal (LC) Optical Phased Array (OPA), also referred to as a spatial light modulator (SLM), is a governing factor that determines the diffraction efficiency (DE) of the LC OPA for high resolution spatial phase modulation. In this article, the fringing field effect in a high resolution LC OPA is studied by accurate modeling the DE of the LC blazed gratings by LC director simulation and Finite Difference Time Domain (FDTD) simulation. Influence factors that contribute significantly to the DE are discussed. Such results provide fundamental understanding for high resolution LC devices.

The 170-kDa glucose-regulated stress protein is an endoplasmic reticulum protein that binds immunoglobulin.

PubMed Central

Lin, H Y; Masso-Welch, P; Di, Y P; Cai, J W; Shen, J W; Subjeck, J R

1993-01-01

Anoxia, glucose starvation, calcium ionophore A23187, EDTA, glucosamine, and several other conditions that adversely affect the function of the endoplasmic reticulum (ER) induce the synthesis of the glucose-regulated class of stress proteins (GRPs). The primary GRPs induced by these stresses migrate at 78 and 94 kDa (GRP78 and GRP94). In addition, another protein of approximately 150-170 kDa (GRP170) has been previously observed and is coordinately induced with GRP78 and GRP94. To characterize this novel stress protein, we have prepared an antisera against purified GRP170. Immunofluorescence, Endoglycosidase H sensitivity, and protease resistance of this protein in microsomes indicates that GRP170 is an ER lumenal glycoprotein retained in a pre-Golgi compartment. Immunoprecipitation of GRP170 with our antibody coprecipitates the GRP78 (also referred to as the B cell immunoglobulin-binding protein) and GRP94 members of this stress protein family in Chinese hamster ovary cells under stress conditions. ATP depletion, by immunoprecipitation in the presence of apyrase, does not affect the interaction between GRP78 and GRP170 but results in the coprecipitation of an unidentified 60-kDa protein. In addition, GRP170 is found to be coprecipitated with immunoglobulin (Ig) in four different B cell hybridomas expressing surface IgM, cytoplasmic Ig light chain only, cytoplasmic Ig heavy chain only, or an antigen specific secreted IgG. In addition, in IgM surface expressing WEHI-231 B cells, anti-IgM coprecipitates GRP78, GRP94, as well as GRP170; antibodies against GRP170 and GRP94 reciprocally coprecipitate GRP94/GRP170 as well as GRP78. Results suggest that this 170-kDa GRP is a retained ER lumenal glycoprotein that is constitutively present and that may play a role in immunoglobulin folding and assembly in conjunction or consecutively with GRP78 and GRP94. Images PMID:8305733

Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated.

PubMed

Reddy, M K; Nair, S; Singh, B N; Mudgil, Y; Tewari, K K; Sopory, S K

2001-01-24

We report the cloning and sequencing of both cDNA and genomic DNA of a 33 kDa chloroplast ribonucleoprotein (33RNP) from pea. The analysis of the predicted amino acid sequence of the cDNA clone revealed that the encoded protein contains two RNA binding domains, including the conserved consensus ribonucleoprotein sequences CS-RNP1 and CS-RNP2, on the C-terminus half and the presence of a putative transit peptide sequence in the N-terminus region. The phylogenetic and multiple sequence alignment analysis of pea chloroplast RNP along with RNPs reported from the other plant sources revealed that the pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RNP and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively. The pea 33RNP was expressed in Escherichia coli and purified to homogeneity. The in vitro import of precursor protein into chloroplasts confirmed that the N-terminus putative transit peptide is a bona fide transit peptide and 33RNP is localized in the chloroplast. The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed that 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDNA and dsDNA. The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated. Sequence analysis of the genomic clone revealed that the gene contains four exons and three introns. We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene.

Fast-response and scattering-free polymer network liquid crystals for infrared light modulators

NASA Astrophysics Data System (ADS)

Fan, Yun-Hsing; Lin, Yi-Hsin; Ren, Hongwen; Gauza, Sebastian; Wu, Shin-Tson

2004-02-01

A fast-response and scattering-free homogeneously aligned polymer network liquid crystal (PNLC) light modulator is demonstrated at λ=1.55 μm wavelength. Light scattering in the near-infrared region is suppressed by optimizing the polymer concentration such that the network domain sizes are smaller than the wavelength. The strong polymer network anchoring assists LC to relax back quickly as the electric field is removed. As a result, the PNLC response time is ˜250× faster than that of the E44 LC mixture except that the threshold voltage is increased by ˜25×.

Identification of an abundant 56 kDa protein implicated in food allergy as granule-bound starch synthase

USDA-ARS?s Scientific Manuscript database

Rice, the staple food of South and East Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16 kDa, 26 kDa, 33 kDa and 56 kDa have been identified as allergens. Re...

Maize 27 kDa gamma-zein is a potential allergen for early weaned pigs.

PubMed

Krishnan, Hari B; Kerley, Monty S; Allee, Gary L; Jang, Sungchan; Kim, Won-Seok; Fu, Chunjiang J

2010-06-23

Soybean and maize are extensively used in animal feed, primarily in poultry, swine, and cattle diets. Soybean meal can affect pig performance in the first few weeks following weaning and elicit specific antibodies in weaned piglets. Though maize is a major component of pig feed, it is not known if any of the maize proteins can elicit immunological response in young pigs. In this study, we have identified a prominent 27 kDa protein from maize as an immunodominant protein in young pigs. This protein, like some known allergens, exhibited resistance to pepsin digestion in vitro. Several lines of evidence identify the immunodominant 27 kDa protein as a gamma-zein, a maize seed storage protein. First, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of different solubility classes of maize seed proteins revealed the presence of an abundant 27 kDa protein in the prolamin (zein) fraction. Antibodies raised against the purified maize 27 kDa gamma-zein also reacted against the same protein recognized by the young pig serum. Additionally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the peptides generated by trypsin digestion of the immunodominant 27 kDa protein showed significant homology to the maize 27 kDa gamma-zein. Since eliminating the allergenic protein will have a great impact on the nutritive value of the maize meal and expand its use in the livestock industry, it will be highly desirable to develop maize cultivars completely lacking the 27 kDa allergenic protein.

30. BEDROOM #3 INTERIOR SHOWING 1 LIGHT OVER 1 LIGHT ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

30. BEDROOM #3 INTERIOR SHOWING 1 LIGHT OVER 1 LIGHT WINDOW ON EAST WALL AND PARTIALLY OPENED DOOR TO WINDOWED CLOSET. VIEW TO EAST. - Big Creek Hydroelectric System, Powerhouse 8, Operator Cottage, Big Creek, Big Creek, Fresno County, CA

Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death.

PubMed

Jiang, Ke; Liu, Min; Lin, Guibin; Mao, Beibei; Cheng, Wei; Liu, Han; Gal, Jozsef; Zhu, Haining; Yuan, Zengqiang; Deng, Wuguo; Liu, Quentin; Gong, Peng; Bi, Xiaolin; Meng, Songshu

2016-05-03

The tumor suppressor Spred2 (Sprouty-related EVH1 domain-2) induces cell death in a variety of cancers. However, the underlying mechanism remains to be elucidated. Here we show that Spred2 induces caspase-independent but autophagy-dependent cell death in human cervical carcinoma HeLa and lung cancer A549 cells. We demonstrate that ectopic Spred2 increased both the conversion of microtubule-associated protein 1 light chain 3 (LC3), GFP-LC3 puncta formation and p62/SQSTM1 degradation in A549 and HeLa cells. Conversely, knockdown of Spred2 in tumor cells inhibited upregulation of autophagosome maturation induced by the autophagy inducer Rapamycin, which could be reversed by the rescue Spred2. These data suggest that Spred2 promotes autophagy in tumor cells. Mechanistically, Spred2 co-localized and interacted with LC3 via the LC3-interacting region (LIR) motifs in its SPR domain. Mutations in the LIR motifs or deletion of the SPR domain impaired Spred2-mediated autophagosome maturation and tumor cell death, indicating that functional LIR is required for Spred2 to trigger tumor cell death. Additionally, Spred2 interacted and co-localized with p62/SQSTM1 through its SPR domain. Furthermore, the co-localization of Spred2, p62 and LAMP2 in HeLa cells indicates that p62 may be involved in Spred2-mediated autophagosome maturation. Inhibition of autophagy using the lysosomal inhibitor chloroquine, reduced Spred2-mediated HeLa cell death. Silencing the expression of autophagy-related genes ATG5, LC3 or p62 in HeLa and A549 cells gave similar results, suggesting that autophagy is required for Spred2-induced tumor cell death. Collectively, these data indicate that Spred2 induces tumor cell death in an autophagy-dependent manner.

Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death

PubMed Central

Lin, Guibin; Mao, Beibei; Cheng, Wei; Liu, Han; Gal, Jozsef; Zhu, Haining; Yuan, Zengqiang; Deng, Wuguo; Liu, Quentin; Gong, Peng; Bi, Xiaolin; Meng, Songshu

2016-01-01

The tumor suppressor Spred2 (Sprouty-related EVH1 domain-2) induces cell death in a variety of cancers. However, the underlying mechanism remains to be elucidated. Here we show that Spred2 induces caspase-independent but autophagy-dependent cell death in human cervical carcinoma HeLa and lung cancer A549 cells. We demonstrate that ectopic Spred2 increased both the conversion of microtubule-associated protein 1 light chain 3 (LC3), GFP-LC3 puncta formation and p62/SQSTM1 degradation in A549 and HeLa cells. Conversely, knockdown of Spred2 in tumor cells inhibited upregulation of autophagosome maturation induced by the autophagy inducer Rapamycin, which could be reversed by the rescue Spred2. These data suggest that Spred2 promotes autophagy in tumor cells. Mechanistically, Spred2 co-localized and interacted with LC3 via the LC3-interacting region (LIR) motifs in its SPR domain. Mutations in the LIR motifs or deletion of the SPR domain impaired Spred2-mediated autophagosome maturation and tumor cell death, indicating that functional LIR is required for Spred2 to trigger tumor cell death. Additionally, Spred2 interacted and co-localized with p62/SQSTM1 through its SPR domain. Furthermore, the co-localization of Spred2, p62 and LAMP2 in HeLa cells indicates that p62 may be involved in Spred2-mediated autophagosome maturation. Inhibition of autophagy using the lysosomal inhibitor chloroquine, reduced Spred2-mediated HeLa cell death. Silencing the expression of autophagy-related genes ATG5, LC3 or p62 in HeLa and A549 cells gave similar results, suggesting that autophagy is required for Spred2-induced tumor cell death. Collectively, these data indicate that Spred2 induces tumor cell death in an autophagy-dependent manner. PMID:27028858

Expression, purification, and characterization of a bifunctional 99-kDa peptidoglycan hydrolase from Pediococcus acidilactici ATCC 8042.

PubMed

García-Cano, Israel; Campos-Gómez, Manuel; Contreras-Cruz, Mariana; Serrano-Maldonado, Carlos Eduardo; González-Canto, Augusto; Peña-Montes, Carolina; Rodríguez-Sanoja, Romina; Sánchez, Sergio; Farrés, Amelia

2015-10-01

Pediococcus acidilactici ATCC 8042 is a lactic acid bacteria that inhibits pathogenic microorganisms such as Staphylococcus aureus through the production of two proteins with lytic activity, one of 110 kDa and the other of 99 kDa. The 99-kDa one has high homology to a putative peptidoglycan hydrolase (PGH) enzyme reported in the genome of P. acidilactici 7_4, where two different lytic domains have been identified but not characterized. The aim of this work was the biochemical characterization of the recombinant enzyme of 99 kDa. The enzyme was cloned and expressed successfully and retains its activity against Micrococcus lysodeikticus. It has a higher N-acetylglucosaminidase activity, but the N-acetylmuramoyl-L-alanine amidase can also be detected spectrophotometrically. The protein was then purified using gel filtration chromatography. Antibacterial activity showed an optimal pH of 6.0 and was stable between 5.0 and 7.0. The optimal temperature for activity was 60 °C, and all activity was lost after 1 h of incubation at 70 °C. The number of strains susceptible to the recombinant 99-kDa enzyme was lower than that susceptible to the mixture of the 110- and 99-kDa PGHs of P. acidilactici, a result that suggests synergy between these two enzymes. This is the first PGH from LAB that has been shown to possess two lytic sites. The results of this study will aid in the design of new antibacterial agents from natural origin that can combat foodborne disease and improve hygienic practices in the industrial sector.

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

PubMed Central

Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

2013-01-01

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies. PMID:23567210

A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions.

PubMed Central

Enrich, C; Bachs, O; Evans, W H

1988-01-01

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3214436

Analytical determination of virginiamycin drug residues in edible porcine tissues by LC-MS with confirmation by LC-MS/MS.

PubMed

Boison, Joe; Lee, Stephen; Gedir, Ron

2009-01-01

A liquid chromatographic-mass spectrometric (LC-MS) method was developed and validated for the determination and confirmation of virginiamycin (VMY) M1 residues in porcine liver, kidney, and muscle tissues at concentrations > or =2 ng/g. Porcine liver, kidney, or muscle tissue is homogenized with methanol-acetonitrile. After centrifugation, the supernatant is diluted with phosphate buffer and cleaned up on a C18 solid-phase extraction cartridge. VMY in the eluate is partitioned into chloroform and the aqueous upper layer is removed by aspiration. After evaporating the chloroform in the residual mixture to dryness, the dried extract is reconstituted in mobile phase and VMY is quantified by LC-MS. Any samples eliciting quantifiable levels of VMY M1 (i.e., at concentrations > or =2 ng/g) are subjected to confirmatory analysis by LC-MSIMS. VMY S1, a minor component of the VMY complex, is monitored but not quantified or confirmed.

Photoaffinity labelling of the cardiac calcium channel. (-)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand.

PubMed

Ferry, D R; Goll, A; Glossmann, H

1987-04-01

The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.

LC3 binding to the scaffolding protein JIP1 regulates processive dynein-driven transport of autophagosomes.

PubMed

Fu, Meng-Meng; Nirschl, Jeffrey J; Holzbaur, Erika L F

2014-06-09

Autophagy is essential for maintaining cellular homeostasis in neurons, where autophagosomes undergo robust unidirectional retrograde transport along axons. We find that the motor scaffolding protein JIP1 binds directly to the autophagosome adaptor LC3 via a conserved LIR motif. This interaction is required for the initial exit of autophagosomes from the distal axon, for sustained retrograde transport along the midaxon, and for autophagosomal maturation in the proximal axon. JIP1 binds directly to the dynein activator dynactin but also binds to and activates kinesin-1 in a phosphorylation-dependent manner. Following JIP1 depletion, phosphodeficient JIP1-S421A rescues retrograde transport, while phosphomimetic JIP1-S421D aberrantly activates anterograde transport. During normal autophagosome transport, residue S421 of JIP1 may be maintained in a dephosphorylated state by autophagosome-associated MKP1 phosphatase. Moreover, binding of LC3 to JIP1 competitively disrupts JIP1-mediated activation of kinesin. Thus, dual mechanisms prevent aberrant activation of kinesin to ensure robust retrograde transport of autophagosomes along the axon. Copyright © 2014 Elsevier Inc. All rights reserved.

Agarose and Polyacrylamide Gel Electrophoresis Methods for Molecular Mass Analysis of 5–500 kDa Hyaluronan

PubMed Central

Bhilocha, Shardul; Amin, Ripal; Pandya, Monika; Yuan, Han; Tank, Mihir; LoBello, Jaclyn; Shytuhina, Anastasia; Wang, Wenlan; Wisniewski, Hans-Georg; de la Motte, Carol; Cowman, Mary K.

2011-01-01

Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5–500 kDa have been investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffers systems were determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition, over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Densitometric scanning of stained gels allowed analysis of the range of molecular masses present in a sample, and calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22 kDa, at sample loads of 0.5 µg (for polyacrylamide) to 2.5 µg (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1-IGD-G2 domains) to a 150 kDa HA standard. PMID:21684248

Accurate LC peak boundary detection for ¹⁶O/¹⁸O labeled LC-MS data.

PubMed

Cui, Jian; Petritis, Konstantinos; Tegeler, Tony; Petritis, Brianne; Ma, Xuepo; Jin, Yufang; Gao, Shou-Jiang S J; Zhang, Jianqiu Michelle

2013-01-01

In liquid chromatography-mass spectrometry (LC-MS), parts of LC peaks are often corrupted by their co-eluting peptides, which results in increased quantification variance. In this paper, we propose to apply accurate LC peak boundary detection to remove the corrupted part of LC peaks. Accurate LC peak boundary detection is achieved by checking the consistency of intensity patterns within peptide elution time ranges. In addition, we remove peptides with erroneous mass assignment through model fitness check, which compares observed intensity patterns to theoretically constructed ones. The proposed algorithm can significantly improve the accuracy and precision of peptide ratio measurements.

Identifying Key Networks Linked to Light-Independent Photoreceptor Degeneration in Visual Arrestin 1 Knockout Mice.

PubMed

Kim, Hwa Sun; Huang, Shun-Ping; Lee, Eun-Jin; Craft, Cheryl Mae

2018-01-01

When visual arrestin 1 (ARR1, S-antigen, 48 KDa protein) was genetically knocked out in mice (original Arr1 -/- , designated Arr1 -/-A ), rod photoreceptors degenerated in a light-dependent manner. Subsequently, a light-independent cone dystrophy was identified with minimal rod death in ARR1 knockout mice (Arr1 -/-A Arr4 +/+ , designated Arr1 -/-B ), which were F2 littermates from breeding the original Arr1 -/-A and cone arrestin knockout 4 (Arr4 -/- ) mice. To resolve the genetic and phenotypic differences between the two ARR1 knockouts, we performed Affymetrix™ exon array analysis to focus on the potential differential gene expression profile and to explore the molecular and cellular pathways leading to this observed susceptibility to cone dystrophy in Arr1 -/-B compared to Arr1 -/-A or control Arr1 +/+ Arr4 +/+ (wild type [WT]). Only in the Arr1 -/-B retina did we observe an up-regulation of retinal transcripts involved in the immune response, inflammatory response and JAK-STAT signaling molecules, OSMRβ and phosphorylation of STAT3. Of these responses, the complement system was significantly higher, and a variety of inflammatory responses by complement regulation and anti-inflammatory cytokine or factors were identified in Arr1 -/-B retinal transcripts. This discovery supports that Arr1 -/-B has a distinct genetic background from Arr1 -/-A that results in alterations in its retinal phenotype leading to susceptibility to cone degeneration induced by inappropriate inflammatory and immune responses.

Transcriptional activation of a 37 kDa ethylene responsive cysteine protease gene, RbCP1, is associated with protein degradation during petal abscission in rose

PubMed Central

Tripathi, Siddharth Kaushal; Singh, Amar Pal; Sane, Aniruddha P.; Nath, Pravendra

2009-01-01

Cysteine proteases play an important role in several developmental processes in plants, particularly those related to senescence and cell death. A cysteine protease gene, RbCP1, has been identified that encodes a putative protein of 357 amino acids and is expressed in the abscission zone (AZ) of petals in rose. The gene was responsive to ethylene in petals, petal abscission zones, leaves, and thalamus. The expression of RbCP1 increased during both ethylene-induced as well as natural abscission and was inhibited by 1-MCP. Transcript accumulation of RbCP1 was accompanied by the appearance of a 37 kDa cysteine protease, a concomitant increase in protease activity and a substantial decrease in total protein content in the AZ of petals. Agro-injection of rose petals with a 2.0 kb region upstream of the RbCP1 gene could drive GUS expression in an abscission zone-specific manner and was blocked by 1-MCP. It is concluded that petal abscission is associated with a decrease in total protein content resulting from rapid transcription of RbCP1 and the expression of a 37 kDa protease. PMID:19346241

Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, R.; Sikorra, S.; Stegmann, C.M.

2009-06-01

Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognitionmore » and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote a-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the a-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.« less

LC3/GABARAP family proteins: autophagy-(un)related functions.

PubMed

Schaaf, Marco B E; Keulers, Tom G; Vooijs, Marc A; Rouschop, Kasper M A

2016-12-01

From yeast to mammals, autophagy is an important mechanism for sustaining cellular homeostasis through facilitating the degradation and recycling of aged and cytotoxic components. During autophagy, cargo is captured in double-membraned vesicles, the autophagosomes, and degraded through lysosomal fusion. In yeast, autophagy initiation, cargo recognition, cargo engulfment, and vesicle closure is Atg8 dependent. In higher eukaryotes, Atg8 has evolved into the LC3/GABARAP protein family, consisting of 7 family proteins [LC3A (2 splice variants), LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2]. LC3B, the most studied family protein, is associated with autophagosome development and maturation and is used to monitor autophagic activity. Given the high homology, the other LC3/GABARAP family proteins are often presumed to fulfill similar functions. Nevertheless, substantial evidence shows that the LC3/GABARAP family proteins are unique in function and important in autophagy-independent mechanisms. In this review, we discuss the current knowledge and functions of the LC3/GABARAP family proteins. We focus on processing of the individual family proteins and their role in autophagy initiation, cargo recognition, vesicle closure, and trafficking, a complex and tightly regulated process that requires selective presentation and recruitment of these family proteins. In addition, functions unrelated to autophagy of the LC3/GABARAP protein family members are discussed.-Schaaf, M. B. E., Keulers, T. G, Vooijs, M. A., Rouschop, K. M. A. LC3/GABARAP family proteins: autophagy-(un)related functions. © FASEB.

Viral Replication Complexes Are Targeted by LC3-Guided Interferon-Inducible GTPases.

PubMed

Biering, Scott B; Choi, Jayoung; Halstrom, Rachel A; Brown, Hailey M; Beatty, Wandy L; Lee, Sanghyun; McCune, Broc T; Dominici, Erin; Williams, Lelia E; Orchard, Robert C; Wilen, Craig B; Yamamoto, Masahiro; Coers, Jörn; Taylor, Gregory A; Hwang, Seungmin

2017-07-12

All viruses with positive-sense RNA genomes replicate on membranous structures in the cytoplasm called replication complexes (RCs). RCs provide an advantageous microenvironment for viral replication, but it is unknown how the host immune system counteracts these structures. Here we show that interferon-gamma (IFNG) disrupts the RC of murine norovirus (MNV) via evolutionarily conserved autophagy proteins and the induction of IFN-inducible GTPases, which are known to destroy the membrane of vacuoles containing bacteria, protists, or fungi. The MNV RC was marked by the microtubule-associated-protein-1-light-chain-3 (LC3) conjugation system of autophagy and then targeted by immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs) upon their induction by IFNG. Further, the LC3 conjugation system and the IFN-inducible GTPases were necessary to inhibit MNV replication in mice and human cells. These data suggest that viral RCs can be marked and antagonized by a universal immune defense mechanism targeting diverse pathogens replicating in cytosolic membrane structures. Copyright © 2017 Elsevier Inc. All rights reserved.

Usefulness of 8 kDa protein of Fasciola hepatica in diagnosis of fascioliasis

PubMed Central

Kim, Kwangsig; Yang, Hyun Jong

2003-01-01

This study was designed to detect and evaluate an antigenicity of low molecular weight proteins of Fasciola hepatica in fascioliasis. Low molecular weight protein of F. hepatica was purified by ammonium sulfate precipitation and Sephacryl S-100 HR gel filtration. The protein obtained was estimated to be 8 kDa on 7.5-15% gradient sodium dodecyl sulfate gel electrophoresis. Immunoblotting studies showed that the 8 kDa protein reacted with human fascioliasis sera, but not other trematodiasis sera. This result suggests that the 8 kDa protein of F. hepatica is one of diagnostic antigens in human fascioliasis without cross-reaction with other human trematodiasis. PMID:12815325

Certification of NIST standard reference material 2389a, amino acids in 0.1 mol/L HCl--quantification by ID LC-MS/MS.

PubMed

Lowenthal, Mark S; Yen, James; Bunk, David M; Phinney, Karen W

2010-05-01

An isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) measurement procedure was developed to accurately quantify amino acid concentrations in National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2389a-amino acids in 0.1 mol/L hydrochloric acid. Seventeen amino acids were quantified using selected reaction monitoring on a triple quadrupole mass spectrometer. LC-MS/MS results were compared to gravimetric measurements from the preparation of SRM 2389a-a reference material developed at NIST and intended for use in intra-laboratory calibrations and quality control. Quantitative mass spectrometry results and gravimetric values were statistically combined into NIST-certified mass fraction values with associated uncertainty estimates. Coefficients of variation (CV) for the repeatability of the LC-MS/MS measurements among amino acids ranged from 0.33% to 2.7% with an average CV of 1.2%. Average relative expanded uncertainty of the certified values including Types A and B uncertainties was 3.5%. Mean accuracy of the LC-MS/MS measurements with gravimetric preparation values agreed to within |1.1|% for all amino acids. NIST SRM 2389a will be available for characterization of routine methods for amino acid analysis and serves as a standard for higher-order measurement traceability. This is the first time an ID LC-MS/MS methodology has been applied for quantifying amino acids in a NIST SRM material.

A Small Molecule Pyrazolo[3,4-d]Pyrimidinone Inhibitor of Zipper-Interacting Protein Kinase Suppresses Calcium Sensitization of Vascular Smooth Muscle.

PubMed

MacDonald, Justin A; Sutherland, Cindy; Carlson, David A; Bhaidani, Sabreena; Al-Ghabkari, Abdulhameed; Swärd, Karl; Haystead, Timothy A J; Walsh, Michael P

2016-01-01

A novel inhibitor of zipper-interacting protein kinase (ZIPK) was used to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pretreatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo[3,4-d]pyrimidinone inhibitor 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)propanamide (HS38) decreased the velocity of contraction (time to reach half-maximal force) induced by the phosphatase inhibitor calyculin A in the presence of Ca(2+) without affecting maximal force development. This effect was reversed following washout of HS38 and correlated with a reduction in the rate of phosphorylation of myosin 20-kDa regulatory light chains (LC20) but not of protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17), prostate apoptosis response-4, or myosin phosphatase-targeting subunit 1 (MYPT1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity toward proviral integrations of Moloney (PIM) virus 3 kinase but not ZIPK, had no effect on calyculin A-induced contraction or protein phosphorylations. We conclude that a pool of constitutively active ZIPK is involved in regulation of vascular smooth muscle contraction through direct phosphorylation of LC20 upon inhibition of myosin light chain phosphatase activity. HS38 also significantly attenuated both phasic and tonic contractile responses elicited by phenylephrine, angiotensin II, endothelin-1, U46619, and K(+)-induced membrane depolarization in the presence of Ca(2+), which correlated with inhibition of phosphorylation of LC20, MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein-coupled receptors that signal through both Gα12/13 and Gαq/11. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Chlorosilane acute inhalation toxicity and development of an LC50 prediction model.

PubMed

Jean, Paul A; Gallavan, Robert H; Kolesar, Gary B; Siddiqui, Waheed H; Oxley, Jon A; Meeks, Robert G

2006-07-01

The acute inhalation toxicity of 10 chlorosilanes was investigated in Fischer 344 rats using a 1-h whole-body vapor inhalation exposure and a 14-day recovery period. The median lethal concentration (LC50(1)) for each material was calculated from the nominal exposure concentrations and mortality. Experimentally derived LC50(1) values for monochlorosilanes (4257-4478 ppm) were greater than those for dichlorosilanes (1785-2092 ppm), which were greater than those for trichlorosilanes (1257-1611 ppm). Apparent was a strong structure-activity relationship (r2 = .97) between chlorine content and LC50(1) value. Estimated LC50(1) values for mono-, di-, and trichlorosilanes were determined to be 3262, 1639, and 1066 ppm, respectively, utilizing this relationship and the lower limit of the 95% prediction interval. The LC50(1) values determined in this series of studies were greater than that reported for hydrogen chloride (3124 ppm), when expressed on a chlorine equivalence basis (3570-5248 ppm), demonstrating that the acute toxicity of these chlorosilanes is similar to or less than that for hydrogen chloride. The good correlation between chlorine content and LC50(1) provides a sound basis for estimation of LC50(1) for chlorosilanes not already evaluated. The use of structure-activity relationships is consistent with the chemical industry and federal agency initiatives to reduce, refine, and/or replace the use of animals in testing without compromising the quality of health and safety assessments.

Wavefront control with a spatial light modulator containing dual-frequency liquid crystal

NASA Astrophysics Data System (ADS)

Gu, Dong-Feng; Winker, Bruce; Wen, Bing; Taber, Don; Brackley, Andrew; Wirth, Allan; Albanese, Marc; Landers, Frank

2004-10-01

A versatile, scalable wavefront control approach based upon proven liquid crystal (LC) spatial light modulator (SLM) technology was extended for potential use in high-energy near-infrared laser applications. The reflective LC SLM module demonstrated has a two-inch diameter active aperture with 812 pixels. Using an ultra-low absorption transparent conductor in the LC SLM, a high laser damage threshold was demonstrated. Novel dual frequency liquid crystal materials and addressing schemes were implemented to achieve fast switching speed (<1ms at 1.31 microns). Combining this LCSLM with a novel wavefront sensing method, a closed loop wavefront controller is being demonstrated. Compared to conventional deformable mirrors, this non-mechanical wavefront control approach offers substantial improvements in speed (bandwidth), resolution, power consumption and system weight/volume.

STS-29 Discovery, Orbiter Vehicle (OV) 103, roll out to KSC LC Pad 39B

NASA Technical Reports Server (NTRS)

1989-01-01

In the early morning hours, STS-29 Discovery, Orbiter Vehicle (OV) 103, mated to the external tank (ET) and solid rocket boosters (SRBs) is rolled out to Kennedy Space Center (KSC) Launch Complex (LC) Pad 39B atop the mobile launcher platform. Trees, shrubs, and a light mist surround the mobile launcher platform as it makes its way to LC Pad 39B. OV-103 will fly on Mission STS-29 scheduled for launch in mid-March. View provided by KSC with alternate KSC number KSC-89PC-50.

Phosphorylation of Tat-interactive protein 60 kDa by protein kinase C epsilon is important for its subcellular localisation.

PubMed

Sapountzi, Vasileia; Logan, Ian R; Nelson, Glyn; Cook, Susan; Robson, Craig N

2008-01-01

Tat-interactive protein 60 kDa is a nuclear acetyltransferase that both coactivates and corepresses transcription factors and has a definitive function in the DNA damage response. Here, we provide evidence that Tat-interactive protein 60 kDa is phosphorylated by protein kinase C epsilon. In vitro, protein kinase C epsilon phosphorylates Tat-interactive protein 60 kDa on at least two sites within the acetyltransferase domain. In whole cells, activation of protein kinase C increases the levels of phosphorylated Tat-interactive protein 60 kDa and the interaction of Tat-interactive protein 60 kDa with protein kinase C epsilon. A phosphomimetic mutant Tat-interactive protein 60 kDa has distinct subcellular localisation compared to the wild-type protein in whole cells. Taken together, these findings suggest that the protein kinase C epsilon phosphorylation sites on Tat-interactive protein 60 kDa are important for its subcellular localisation. Regulation of the subcellular localisation of Tat-interactive protein 60 kDa via phosphorylation provides a novel means of controlling Tat-interactive protein 60 kDa function.

Double-layered liquid crystal light shutter for control of absorption and scattering of the light incident to a transparent display device

NASA Astrophysics Data System (ADS)

Huh, Jae-Won; Yu, Byeong-Hun; Shin, Dong-Myung; Yoon, Tae-Hoon

2015-03-01

Recently, a transparent display has got much attention as one of the next generation display devices. Especially, active studies on a transparent display using organic light-emitting diodes (OLEDs) are in progress. However, since it is not possible to obtain black color using a transparent OLED, it suffers from poor visibility. This inevitable problem can be solved by using a light shutter. Light shutter technology can be divided into two types; light absorption and scattering. However, a light shutter based on light absorption cannot block the background image perfectly and a light shutter based on light scattering cannot provide black color. In this work we demonstrate a light shutter using two liquid crystal (LC) layers, a light absorption layer and a light scattering layer. To realize a light absorption layer and a light scattering layer, we use the planar state of a dye-doped chiral nematic LC (CNLC) cell and the focal-conic state of a long-pitch CNLC cell, respectively. The proposed light shutter device can block the background image perfectly and show black color. We expect that the proposed light shutter can increase the visibility of a transparent display.

Identification and molecular characterization of 48 kDa calcium binding protein as calreticulin from finger millet (Eleusine coracana) using peptide mass fingerprinting and transcript profiling.

PubMed

Singh, Manoj; Metwal, Mamta; Kumar, Vandana A; Kumar, Anil

2016-01-30

Attempts were made to identify and characterize the calcium binding proteins (CaBPs) in grain filling stages of finger millet using proteomics, bioinformatics and molecular approaches. A distinctly observed blue color band of 48 kDa stained by Stains-all was eluted and analyzed as calreticulin (CRT) using nano liquid chromatography-tandem mass spectrometry (nano LC-MS). Based on the top hits of peptide mass fingerprinting results, conserved primers were designed for isolation of the CRT gene from finger millet using calreticulin sequences of different cereals. The deduced nucleotide sequence analysis of 600 bp amplicon showed up to 91% similarity with CRT gene(s) of rice and other plant species and designated as EcCRT1. Transcript profiling of EcCRT1 showed different levels of relative expression at different stages of developing spikes. The higher expression of EcCRT1 transcripts and protein were observed in later stages of developing spikes which might be due to greater translational synthesis of EcCRT1 protein during seed maturation in finger millet. Preferentially higher synthesis of this CaBP during later stages of grain filling may be responsible for the sequestration of calcium in endoplasmic reticulum of finger millet grains. © 2015 Society of Chemical Industry.

The analysis Arabidopsis thaliana overexpressing a 14kDa self-folding protein [abstract

USDA-ARS?s Scientific Manuscript database

A recent study in banana identified a 14kDa protein that has been hypothesized to function in regulating the nucleation and growth of the needle-shaped crystals of calcium oxalate that accumulate within the tissues of this plant. To gain further insight in to the functional role of this 14 kDa prote...

Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD.

PubMed

Goode, Alice; Butler, Kevin; Long, Jed; Cavey, James; Scott, Daniel; Shaw, Barry; Sollenberger, Jill; Gell, Christopher; Johansen, Terje; Oldham, Neil J; Searle, Mark S; Layfield, Robert

2016-07-02

Growing evidence implicates impairment of autophagy as a candidate pathogenic mechanism in the spectrum of neurodegenerative disorders which includes amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS-FTLD). SQSTM1, which encodes the autophagy receptor SQSTM1/p62, is genetically associated with ALS-FTLD, although to date autophagy-relevant functional defects in disease-associated variants have not been described. A key protein-protein interaction in autophagy is the recognition of a lipid-anchored form of LC3 (LC3-II) within the phagophore membrane by SQSTM1, mediated through its LC3-interacting region (LIR), and notably some ALS-FTLD mutations map to this region. Here we show that although representing a conservative substitution and predicted to be benign, the ALS-associated L341V mutation of SQSTM1 is defective in recognition of LC3B. We place our observations on a firm quantitative footing by showing the L341V-mutant LIR is associated with a ∼3-fold reduction in LC3B binding affinity and using protein NMR we rationalize the structural basis for the effect. This functional deficit is realized in motor neuron-like cells, with the L341V mutant EGFP-mCherry-SQSTM1 less readily incorporated into acidic autophagic vesicles than the wild type. Our data supports a model in which the L341V mutation limits the critical step of SQSTM1 recruitment to the phagophore. The oligomeric nature of SQSTM1, which presents multiple LIRs to template growth of the phagophore, potentially gives rise to avidity effects which amplify the relatively modest impact of any single mutation on LC3B binding. Over the lifetime of a neuron, impaired autophagy could expose a vulnerability, which ultimately tips the balance from cell survival toward cell death.

Voltage- and temperature- controlled LC:PDMS waveguide channels

NASA Astrophysics Data System (ADS)

Rutkowska, Katarzyna A.; Asquini, Rita; d'Alessandro, Antonio

2017-08-01

In this paper, we present our studies on electrical and thermal tuning of light propagation in waveguide channels, made for the scope from a polydimethylsiloxane (PDMS) substrate infiltrated with nematic liquid crystal (LC). We demonstrated, via numerical simulations, the changes of the waveguide optical parameters when solicited by temperature changes or electric fields. Moreover, the paper goes through the fabrication process of a waveguide channel sample and its characterization, as well as some preliminary experimental trials of sputtering indium tin oxide (ITO) and chromium layers on PDMS substrate to obtain flat electrodes.

Single-Prolonged-Stress-Induced Changes in Autophagy-Related Proteins Beclin-1, LC3, and p62 in the Medial Prefrontal Cortex of Rats with Post-traumatic Stress Disorder.

PubMed

Zheng, Shilei; Han, Fang; Shi, Yuxiu; Wen, Lili; Han, Dan

2017-05-01

Autophagy, or type II programmed cell death, plays a crucial role in many nervous system diseases. However, few studies have examined the role of autophagy in post-traumatic stress disorder (PTSD), and the mechanisms underlying PTSD are poorly understood. The objective of this research was to explore the expression of three important autophagy-related proteins, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and p62/SQSTM1 (p62), in the medial prefrontal cortex (mPFC) of an animal model of PTSD to identify changes in autophagic activity during PTSD pathogenesis. PTSD was induced in rats by exposure to a single-prolonged stress (SPS). The Morris water maze was used to assess cognitive changes in rats from the SPS and control groups. Transmission electron microscopy (TEM) was employed to observe mPFC morphological changes. Immunohistochemistry, immunofluorescence, and Western blotting techniques were used to detect expression of Beclin-1, LC3, and p62 in the mPFC. The Morris water maze test results showed that the escape latency time was increased and that the percent time in the target quadrant was decreased in the SPS group compared with that in the control group. Numerous visible autolysosomes in mPFC neurons were observed using TEM after SPS stimulation. Compared with that in the control group, the expression of Beclin-1 and the LC3-II/I ratio significantly decreased at 1 day, then increased and peaked at 7 days, and slightly decreased at 14 days after SPS stimulation, whereas the converse was found for p62 expression. In conclusion, dysregulation of autophagic activity in the mPFC may play a crucial role in PTSD pathogenesis.

Oleosins (24 and 18 kDa) are hydrolyzed not only in extracted soybean oil bodies but also in soybean germination.

PubMed

Chen, Yeming; Zhao, Luping; Cao, Yanyun; Kong, Xiangzhen; Hua, Yufei

2014-01-29

After oil bodies (OBs) were extracted from ungerminated soybean by pH 6.8 extraction, it was found that 24 and 18 kDa oleosins were hydrolyzed in the extracted OBs, which contained many OB extrinsic proteins (i.e., lipoxygenase, β-conglycinin, γ-conglycinin, β-amylase, glycinin, Gly m Bd 30K (Bd 30K), and P34 probable thiol protease (P34)) as well as OB intrinsic proteins. In this study, some properties (specificity, optimal pH and temperature) of the proteases of 24 and 18 kDa oleosins and the oleosin hydrolysis in soybean germination were examined, and the high relationship between Bd 30K/P34 and the proteases was also discussed. The results showed (1) the proteases were OB extrinsic proteins, which had high specificity to hydrolyze 24 and 18 kDa oleosins, and cleaved the specific peptide bonds to form limited hydrolyzed products; (2) 24 and 18 kDa oleosins were not hydrolyzed in the absence of Bd 30K and P34 (or some Tricine-SDS-PAGE undetectable proteins); (3) the protease of 24 kDa oleosin had strong resistance to alkaline pH while that of 18 kDa oleosin had weak resistance to alkaline pH, and Bd 30K and P34, resolved into two spots on two-dimensional electrophoresis gel, also showed the same trend; (4) 16 kDa oleosin as well as 24 and 18 kDa oleosins were hydrolyzed in soybean germination, and Bd 30K and P34 were always contained in the extracted OBs from germinated soybean even when all oleosins were hydrolyzed; (5) the optimal temperature and pH of the proteases were respectively determined as in the ranges of 35-50 °C and pH 6.0-6.5, while 60 °C or pH 11.0 could denature them.

Alterations of apoptosis and autophagy in developing brain of rats with epilepsy: Changes in LC3, P62, Beclin-1 and Bcl-2 levels.

PubMed

Li, Qinrui; Han, Ying; Du, Junbao; Jin, Hongfang; Zhang, Jing; Niu, Manman; Qin, Jiong

2018-05-01

Current studies have indicated that apoptotic and autophagic signaling pathways are triggered by epileptic seizures, but the precise roles of these processes in epilepsy-induced neuronal loss remain unclear. Identifying a concrete molecular mechanism may help researchers develop relevant epilepsy therapies that are more effective than existing treatments. Autophagy is a type of conserved degradation that contributes to cellular homeostasis. The involved signaling pathways allow us to observe alterations in autophagy and apoptosis during epileptic seizures over time. This study investigated the time-dependent changes in autophagy, apoptosis and neuronal morphology in developing brain of epilepsy model rats. At 48h after epileptic seizure onset, the number of neurons in neocortex decreased, and the number of apoptotic cells in neocortex increased. The ratio of microtubule-associated protein 1 light chain 3 (LC3) II to LC3 I and Beclin-1 protein levels increased from 12h to 48h after epileptic seizure onset. P62 protein and Bcl-2 protein levels decreased from 24h to 48h after epileptic seizure onset. The changes in the levels of these autophagy and apoptosis markers indicate that autophagy starts before apoptosis in rats with epilepsy, demonstrating a potential role of autophagy in epilepsy-induced neuronal loss in developing brain. Copyright © 2017. Published by Elsevier B.V.

Light distribution modulated diffuse reflectance spectroscopy.

PubMed

Huang, Pin-Yuan; Chien, Chun-Yu; Sheu, Chia-Rong; Chen, Yu-Wen; Tseng, Sheng-Hao

2016-06-01

Typically, a diffuse reflectance spectroscopy (DRS) system employing a continuous wave light source would need to acquire diffuse reflectances measured at multiple source-detector separations for determining the absorption and reduced scattering coefficients of turbid samples. This results in a multi-fiber probe structure and an indefinite probing depth. Here we present a novel DRS method that can utilize a few diffuse reflectances measured at one source-detector separation for recovering the optical properties of samples. The core of innovation is a liquid crystal (LC) cell whose scattering property can be modulated by the bias voltage. By placing the LC cell between the light source and the sample, the spatial distribution of light in the sample can be varied as the scattering property of the LC cell modulated by the bias voltage, and this would induce intensity variation of the collected diffuse reflectance. From a series of Monte Carlo simulations and phantom measurements, we found that this new light distribution modulated DRS (LDM DRS) system was capable of accurately recover the absorption and scattering coefficients of turbid samples and its probing depth only varied by less than 3% over the full bias voltage variation range. Our results suggest that this LDM DRS platform could be developed to various low-cost, efficient, and compact systems for in-vivo superficial tissue investigation.

Light distribution modulated diffuse reflectance spectroscopy

PubMed Central

Huang, Pin-Yuan; Chien, Chun-Yu; Sheu, Chia-Rong; Chen, Yu-Wen; Tseng, Sheng-Hao

2016-01-01

Typically, a diffuse reflectance spectroscopy (DRS) system employing a continuous wave light source would need to acquire diffuse reflectances measured at multiple source-detector separations for determining the absorption and reduced scattering coefficients of turbid samples. This results in a multi-fiber probe structure and an indefinite probing depth. Here we present a novel DRS method that can utilize a few diffuse reflectances measured at one source-detector separation for recovering the optical properties of samples. The core of innovation is a liquid crystal (LC) cell whose scattering property can be modulated by the bias voltage. By placing the LC cell between the light source and the sample, the spatial distribution of light in the sample can be varied as the scattering property of the LC cell modulated by the bias voltage, and this would induce intensity variation of the collected diffuse reflectance. From a series of Monte Carlo simulations and phantom measurements, we found that this new light distribution modulated DRS (LDM DRS) system was capable of accurately recover the absorption and scattering coefficients of turbid samples and its probing depth only varied by less than 3% over the full bias voltage variation range. Our results suggest that this LDM DRS platform could be developed to various low-cost, efficient, and compact systems for in-vivo superficial tissue investigation. PMID:27375931

[Comparison of surface light scattering of acrylic intraocular lenses made by lathe-cutting and cast-molding methods--long-term observation and experimental study].

PubMed

Nishihara, Hitoshi; Ayaki, Masahiko; Watanabe, Tomiko; Ohnishi, Takeo; Kageyama, Toshiyuki; Yaguchi, Shigeo

2004-03-01

To compare the long-term clinical and experimental results of soft acrylic intraocular lenses(IOLs) manufactured by the lathe-cut(LC) method and by the cast-molding(CM) method. This was a retrospective study of 20 patients(22 eyes) who were examined in a 5- and 7-year follow-up study. Sixteen eyes were implanted with polyacrylic IOLs manufactured by the LC method and 6 eyes were implanted with polyacrylic IOLs manufactured by the CM method. Postoperative measurements included best corrected visual acuity, contrast sensitivity, biomicroscopic examination, and Scheimpflug slit-lamp images to evaluate surface light scattering. Scanning electron microscopy and three-dimensional surface analysis were conducted. At 7 years, the mean visual acuity was 1.08 +/- 0.24 (mean +/- standard deviation) in the LC group and 1.22 +/- 0.27 in the CM group. Surface light-seatter was 12.0 +/- 4.0 computer compatible tapes(CCT) in the LC group and 37.4 +/- 5.4 CCT in the CM group. Mean surface roughness was 0.70 +/- 0.07 nm in the LC group and 6.16 +/- 0.97 nm in the CM group. Acrylic IOLs manufactured by the LC method are more stable in long-termuse.

Morphing dynamics in light-triggered LC polymers (Conference Presentation)

NASA Astrophysics Data System (ADS)

Broer, Dirk J.

2017-02-01

Polymers that can change shape or surface topography in response to a trigger have a wide application potential varying from micro-robotics to avionics. Preferably this morphing proceeds fast and reversibly. We developed new morphing principles based on in-situ photopolymerized liquid crystal networks and on hybrid low molecular weight liquid crystals and liquid crystal networks. Commonly the triggers are temperature, light, pH or the presence of chemicals or other moisture. In the lecture we will focus on UV actuation and demonstrate that by accurate positioning of molecules over all three dimensions of a thin film or coating, the deformation figures can be pre-engineered. They can vary from simple gratings to very complex such as fingerprints that can be switched between off (flat surface) and on (corrugated surface) by light. The underlying principles are based on photo-induced changes in the degree of order of liquid crystal polymer networks and the accompanying changes in density by the formation of free volume. The surfaces can be switched with frequencies of the order of 0.1 Hz. In the lecture we will discuss several methods to fabricate the responsive layers as well as some of the most eye-catching properties. Also the mechanism of free volume generation will be addressed in terms of molecular dynamics and resonance.

Formation of the 67-kDa laminin receptor by acylation of the precursor.

PubMed

Butò, S; Tagliabue, E; Ardini, E; Magnifico, A; Ghirelli, C; van den Brûle, F; Castronovo, V; Colnaghi, M I; Sobel, M E; Ménard, S

1998-06-01

Even though the involvement of the 67-kDa laminin receptor (67LR) in tumor invasiveness has been clearly demonstrated, its molecular structure remains an open problem, since only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated so far. A pool of recently obtained monoclonal antibodies directed against the recombinant 37LRP molecule was used to investigate the processing that leads to the formation of the 67-kDa molecule. In soluble extracts of A431 human carcinoma cells, these reagents recognize the precursor molecule as well as the mature 67LR and a 120-kDa molecule. The recovery of these proteins was found to be strikingly dependent upon the cell solubilization conditions: the 67LR is soluble in NP-40-lysis buffer whereas the 37LRP is NP-40-insoluble. Inhibition of 67LR formation by cerulenin indicates that acylation is involved in the processing of the receptor. It is likely a palmitoylation process, as indicated by sensitivity of NP-40-soluble extracts to hydroxylamine treatment. Immunoblotting assays performed with a polyclonal serum directed against galectin3 showed that both the 67- and the 120-kDa proteins carry galectin3 epitopes whereas the 37LRP does not. These data suggest that the 67LR is a heterodimer stabilized by strong intramolecular hydrophobic interactions, carried by fatty acids bound to the 37LRP and to a galectin3 cross-reacting molecule.

A Pedagogical Measurement of the Velocity of Light

ERIC Educational Resources Information Center

Tyler, Charles E.

1969-01-01

Describes an inexpensive, easily constructed device for demonstrating that the speed of light is finite, and for measuring its value. The main components are gallium arsenide light emitting diodes, a light pulser, transistors, and an oscilloscope. Detailed instructions of procedure and experimental results are given. (LC)

Therapeutic Approaches for Botulinum Intoxication Targeting Degradation of the Light Chain

DTIC Science & Technology

2014-06-01

protein and producing adequate amounts for in vitro testing. 15. SUBJECT TERMS- Botulinum toxin , ubiquitin, chimeric toxin light chains, LcA, LcE...that confer stability to LCs of botulinum toxin can be assessed by mutation of dileucine residues and systematic deletion of residues from LcA-LcE...cells. So What? Currently, there is no cure for botulinum poisoning once the toxin has entered a neuron. Moreover, the half-life of BoNT/A is very

33. INTERIOR OF BEDROOM NO. 4 SHOWING 1LIGHT OVER 1LIGHT, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

33. INTERIOR OF BEDROOM NO. 4 SHOWING 1-LIGHT OVER 1-LIGHT, DOUBLE-HUNG WINDOWS. VIEW TO NORTHWEST. - Bishop Creek Hydroelectric System, Plant 6, Cashbaugh-Kilpatrick House, Bishop Creek, Bishop, Inyo County, CA

The change of nuclear LC3 distribution in acute myeloid leukemia cells.

PubMed

Guo, Wenjian; Jin, Jingrui; Pan, Jiajia; Yao, Rongxing; Li, Xia; Huang, Xin; Ma, Zhixing; Huang, Sujuan; Yan, Xiao; Jin, Jie; Dong, Aishu

2018-05-09

Making sure the change of nuclear LC3 distribution in the autophagy of acute myeloid leukemia (AML) cell and finding out the regulation mechanism may lead to a breakthrough for killing AML cells. Western blots were performed to assess the expression of autophagy proteins. Changes in the LC3 distribution were monitored by immunofluorescence assays together with western blots, and the expression levels of Sirt1, DOR, Beclin1, HMGB1, and AMPK mRNA were detected via fluorescent quantitative PCR. The effects of Sirt1 and DOR on cell proliferation and survival were analyzed by MTT, flow cytometry, and western blotting assays. We found that treating AML cells with Ara-c or Sorafenib resulted in autophagy enhancement, and when autophagy was enhanced, nuclear LC3 moved into the cytoplasm. Notably, when autophagy was inhibited by blocking the nuclear LC3 shift, the cytotoxicity of drugs was enhanced. Our results also identified Sirt1 and DOR as regulatory molecules for the observed nuclear LC3 shift, and these molecules further affected the expression of Beclin1, HMGB1, and AMPK. Our results suggest the distribution of nuclear LC3 can be a novel way for further studying death of AML cells,and the regulatory molecules may be new targets for treating AML. Copyright © 2018 Elsevier Inc. All rights reserved.

Fermentation, degradation and microbial nitrogen partitioning for three forage colour phenotypes within anthocyanidin-accumulating Lc-alfalfa progeny.

PubMed

Jonker, Arjan; Gruber, Margaret Y; Wang, Yuxi; Narvaez, Nelmy; Coulman, Bruce; McKinnon, John J; Christensen, David A; Azarfar, Arash; Yu, Peiqiang

2012-08-30

Alfalfa has the disadvantage of having a rapid initial rate of protein degradation, which results in pasture bloat, low efficiency of protein utilisation and excessive nitrogen (N) pollution into the environment for cattle. Introducing a gene that stimulates the accumulation of monomeric/polymeric anthocyanidins might reduce the ruminal protein degradation rate (by fixing protein and/or direct interaction with microbes) and additionally reduce methane emission. The objectives of this study were to evaluate in vitro fermentation, degradation and microbial N partitioning of three forage colour phenotypes (green, light purple-green (LPG) and purple-green (PG)) within newly developed Lc-progeny and to compare them with those of parental green non-transgenic (NT) alfalfa. PG-Lc accumulated more anthocyanidin compared with Green-Lc (P < 0.05), with LPG-Lc intermediate. Volatile fatty acids and potentially degradable dry matter (DM) and N were similar among the four phenotypes. Gas, methane and ammonia accumulation rates were slower for the two purple-Lc phenotypes compared with NT-alfalfa (P < 0.05), while Green-Lc was intermediate. Effective degradable DM and N were lower in the three Lc-phenotypes (P < 0.05) compared with NT-alfalfa. Anthocyanidin concentration was negatively correlated (P < 0.05) with gas and methane production rates and effective degradability of DM and N. The Lc-alfalfa phenotypes accumulated anthocyanidin. Fermentation and degradation parameters indicated a reduced rate of fermentation and effective degradability for both purple anthocyanidin-accumulating Lc-alfalfa phenotypes compared with NT-alfalfa. Copyright © 2012 Society of Chemical Industry.

Translocation of an 89-kDa periplasmic protein is associated with Holospora infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwatani, Koichi; Dohra, Hideo; Lang, B. Franz

2005-12-02

The symbiotic bacterium Holospora obtusa infects the macronucleus of the ciliate Paramecium caudatum. After ingestion by its host, an infectious form of Holospora with an electron-translucent tip passes through the host digestive vacuole and penetrates the macronuclear envelope with this tip. To investigate the underlying molecular mechanism of this process, we raised a monoclonal antibody against the tip-specific 89-kDa protein, sequenced this partially, and identified the corresponding complete gene. The deduced protein sequence carries two actin-binding motifs. Indirect immunofluorescence microscopy shows that during escape from the host digestive vacuole, the 89-kDa proteins translocates from the inside to the outside ofmore » the tip. When the bacterium invades the macronucleus, the 89-kDa protein is left behind at the entry point of the nuclear envelope. Transmission electron microscopy shows the formation of fine fibrous structures that co-localize with the antibody-labeled regions of the bacterium. Our findings suggest that the 89-kDa protein plays a role in Holospora's escape from the host digestive vacuole, the migration through the host cytoplasm, and the invasion into the macronucleus.« less

Phenotyping polyclonal kappa and lambda light chain molecular mass distributions in patient serum using mass spectrometry.

PubMed

Barnidge, David R; Dasari, Surendra; Ramirez-Alvarado, Marina; Fontan, Adrian; Willrich, Maria A V; Tschumper, Renee C; Jelinek, Diane F; Snyder, Melissa R; Dispenzieri, Angela; Katzmann, Jerry A; Murray, David L

2014-11-07

We previously described a microLC-ESI-Q-TOF MS method for identifying monoclonal immunoglobulins in serum and then tracking them over time using their accurate molecular mass. Here we demonstrate how the same methodology can be used to identify and characterize polyclonal immunoglobulins in serum. We establish that two molecular mass distributions observed by microLC-ESI-Q-TOF MS are from polyclonal kappa and lambda light chains using a combination of theoretical molecular masses from gene sequence data and the analysis of commercially available purified polyclonal IgG kappa and IgG lambda from normal human serum. A linear regression comparison of kappa/lambda ratios for 74 serum samples (25 hypergammaglobulinemia, 24 hypogammaglobulinemia, 25 normal) determined by microflowLC-ESI-Q-TOF MS and immunonephelometry had a slope of 1.37 and a correlation coefficient of 0.639. In addition to providing kappa/lambda ratios, the same microLC-ESI-Q-TOF MS analysis can determine the molecular mass for oligoclonal light chains observed above the polyclonal background in patient samples. In 2 patients with immune disorders and hypergammaglobulinemia, we observed a skewed polyclonal molecular mass distribution which translated into biased kappa/lambda ratios. Mass spectrometry provides a rapid and simple way to combine the polyclonal kappa/lambda light chain abundance ratios with the identification of dominant monoclonal as well as oligoclonal light chain immunoglobulins. We anticipate that this approach to evaluating immunoglobulin light chains will lead to improved understanding of immune deficiencies, autoimmune diseases, and antibody responses.

Definitive screening design enables optimization of LC-ESI-MS/MS parameters in proteomics.

PubMed

Aburaya, Shunsuke; Aoki, Wataru; Minakuchi, Hiroyoshi; Ueda, Mitsuyoshi

2017-12-01

In proteomics, more than 100,000 peptides are generated from the digestion of human cell lysates. Proteome samples have a broad dynamic range in protein abundance; therefore, it is critical to optimize various parameters of LC-ESI-MS/MS to comprehensively identify these peptides. However, there are many parameters for LC-ESI-MS/MS analysis. In this study, we applied definitive screening design to simultaneously optimize 14 parameters in the operation of monolithic capillary LC-ESI-MS/MS to increase the number of identified proteins and/or the average peak area of MS1. The simultaneous optimization enabled the determination of two-factor interactions between LC and MS. Finally, we found two parameter sets of monolithic capillary LC-ESI-MS/MS that increased the number of identified proteins by 8.1% or the average peak area of MS1 by 67%. The definitive screening design would be highly useful for high-throughput analysis of the best parameter set in LC-ESI-MS/MS systems.

Characterisation of the ester-substituted products of the reaction of p-t-butyl calix[4]arene and ethyl bromoacetate using LC-UV-MS and LC-DAD.

PubMed

McMahon, Gillian; Wall, Rachel; Nolan, Kieran; Diamond, Dermot

2002-07-19

A series of derivatisation reactions between p-t-butyl calix[4]arene and ethyl bromoacetate were carried out in order to prepare 1,3 diester substituted calix[4]arene. Mass spectral data, obtained from direct injection of samples, indicated that the reactions were rich in the desired product. Since the ultra violet (UV) spectra of the desired product and possible impurities are very similar, liquid chromatography (LC) chromatographic data seemed to corroborate these results. However, when on-line LC-UV-MS was carried out and each LC peak subjected to MS analysis as it eluted, a very different picture emerged. It was found that many of these reactions actually contained high levels of the monoester product which, having less affinity for sodium in the MS, is therefore seriously underestimated in any direct injection assay. LC-diode array detection (DAD) methods were also used to help successfully identify and characterise the compounds being formed in these complex reactions. The overall results obtained in this paper allowed the optimal reaction conditions to be determined for this reaction. LC-MS analysis of the chromatographic peaks also identified the presence of two isomers of the diester substituted calix[4]arene (1,3 and 1,2 diesters). The combination of LC and UV/MS detection is required for accurate analysis of the products of such reactions.

Validation and use of three complementary analytical methods (LC-FLS, LC-MS/MS and ICP-MS) to evaluate the pharmacokinetics, biodistribution and stability of motexafin gadolinium in plasma and tissues.

PubMed

Miles, Dale R; Mesfin, Mimi; Mody, Tarak D; Stiles, Mark; Lee, Jean; Fiene, John; Denis, Bernie; Boswell, Garry W

2006-05-01

Liquid chromatography-fluorescence (LC-FLS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and inductively coupled plasma-mass spectrometry (ICP-MS) methods were developed and validated for the evaluation of motexafin gadolinium (MGd, Xcytrin) pharmacokinetics and biodistribution in plasma and tissues. The LC-FLS method exhibited the greatest sensitivity (0.0057 microg mL(-1)), and was used for pharmacokinetic, biodistribution, and protein binding studies with small sample sizes or low MGd concentrations. The LC-MS/MS method, which exhibited a short run time and excellent selectivity, was used for routine clinical plasma sample analysis. The ICP-MS method, which measured total Gd, was used in conjunction with LC methods to assess MGd stability in plasma. All three methods were validated using human plasma. The LC-FLS method was also validated using plasma, liver and kidneys from mice and rats. All three methods were shown to be accurate, precise and robust for each matrix validated. For three mice, the mean (standard deviation) concentration of MGd in plasma/tissues taken 5 hr after dosing with 23 mg kg(-1) MGd was determined by LC-FLS as follows: plasma (0.025+/-0.002 microg mL(-1)), liver (2.89+/-0.45 microg g(-1)), and kidney (6.09+/-1.05 microg g(-1)). Plasma samples from a subset of patients with brain metastases from extracranial tumors were analyzed using both LC-MS/MS and ICP-MS methods. For a representative patient, > or = 90% of the total Gd in plasma was accounted for as MGd over the first hour post dosing. By 24 hr post dosing, 63% of total Gd was accounted for as MGd, indicating some metabolism of MGd.

Light scattering by a nematic liquid crystal droplet: Wentzel–Kramers–Brillouin approximation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loiko, V. A., E-mail: loiko@dragon.bas-net.by; Konkolovich, A. V.; Miskevich, A. A.

2016-01-15

Light scattering by an optically anisotropic liquid crystal (LC) droplet of a nematic in an isotropic polymer matrix is considered in the Wentzel–Kramers–Brillouin (WKB) approximation. General relations are obtained for elements of the amplitude matrix of light scattering by a droplet of arbitrary shape and for the structure of the director field. Analytic expressions for the amplitude matrices are derived for spherical LC droplets with a uniformly oriented structure of local optical axes for strictly forward and strictly backward scattering. The efficiency factors of extinction and backward scattering for a spherical nonabsorbing LC droplet depending on the LC optical anisotropy,more » refractive index of the polymer, illumination conditions, and orientation of the optical axis of the droplet are analyzed. Verification of the obtained solutions has been performed.« less

Recent advances in IR liquid crystal spatial light modulators

NASA Astrophysics Data System (ADS)

Peng, Fenglin; Twieg, Robert J.; Wu, Shin-Tson

2015-09-01

Liquid crystal (LC) is an amazing class of electro-optic media; its applications span from visible to infrared, millimeter wave, and terahertz regions. In the visible and short-wavelength infrared (SWIR) regions, most LCs are highly transparent. However, to extend the electro-optic application of LCs into MWIR and LWIR, several key technical challenges have to be overcome: (1) low absorption loss, (2) high birefringence, (3) low operation voltage, and (4) fast response time. In the MWIR and LWIR regions, several fundamental molecular vibration bands and overtones exist, which contribute to high absorption loss. The absorbed light turns to heat and then alters the birefringence locally, which in turns causes spatially non-uniform phase modulation. To suppress the optical loss, several approaches have been investigated: (1) Employing thin cell gap by choosing a high birefringence LC mixture; (2) Shifting the absorption bands outside the spectral region of interest by deuteration, fluorination, or chlorination; (3) Reducing the overtone absorption by using a short alkyl chain. In this paper, we report some recently developed chlorinated LC compounds and mixtures with low absorption loss in the SWIR and MWIR regions. To achieve fast response time, we demonstrated a polymer network liquid crystal with 2π phase change at MWIR and response time less than 5 ms. Approaches to extend such a liquid crystal spatial light modulator to long-wavelength infrared will be discussed.

The 33.1 kDa Excretory/secretory Protein Produced by Toxocara canis Larvae Serves as a Potential Common Biomarker for Serodiagnosis of Toxocariasis in Paratenic Animals and Human.

PubMed

Nguyen, Huu-Hung; Vo, Doan-Trung; Thai, Thi-Tuyet-Trinh; LE, Thi-Thanh-Thao; LE, Thanh-Dong; Hoang, Nghia-Son

2017-01-01

Toxocariasis is a prevalent zoonosis disease caused by the closely related nematode species Toxocara canis and Toxocara cati which parasitise Canidae and Felidae respectively. In paratenic hosts, larvae of these worms cause multiple organ damage. However, how these paratenic hosts response to these worms and whether any common biomarker can be applied for diagnosis are still unclear. Excreted/secreted (E/S) antigens were prepared by culture of T. canis larvae in vitro. Using a western blot (WB) assay the humoral IgG responses, induced by Toxocara spp. larvae to the worm's E/S antigens in different infected hosts including mice, rabbits and human, were examined. In a mouse model of toxocariasis, intraperitoneal injection of T. canis larvae induces inflammatory leukocyte accumulation in the liver and the lungs but not in the brain, although a remarkable number of larvae were detected in this organ. Mice and rabbits responded differently to Toxocara spp. resulting in distinct heterogenous WB band patterns. Mice and rabbits both responded to a 33.1 kDa E/S constituent that turned out to be the most sensitive protein for serodiagnosis. Sera from human toxocariasis patients showed heterogenous WB band patterns similar to those observed in rabbits and all responded to the 33.1 kDa band. 33.1 kDa E/S protein can be considered as a critical common biomarker for toxocariasis immuno-diagnosis in both paratenic animals and human and its specificity requires further investigation.

The 33.1 kDa Excretory/secretory Protein Produced by Toxocara canis Larvae Serves as a Potential Common Biomarker for Serodiagnosis of Toxocariasis in Paratenic Animals and Human

PubMed Central

NGUYEN, Huu-Hung; VO, Doan-Trung; THAI, Thi-Tuyet-Trinh; LE, Thi-Thanh-Thao; LE, Thanh-Dong; HOANG, Nghia-Son

2017-01-01

Background: Toxocariasis is a prevalent zoonosis disease caused by the closely related nematode species Toxocara canis and Toxocara cati which parasitise Canidae and Felidae respectively. In paratenic hosts, larvae of these worms cause multiple organ damage. However, how these paratenic hosts response to these worms and whether any common biomarker can be applied for diagnosis are still unclear. Methods: Excreted/secreted (E/S) antigens were prepared by culture of T. canis larvae in vitro. Using a western blot (WB) assay the humoral IgG responses, induced by Toxocara spp. larvae to the worm’s E/S antigens in different infected hosts including mice, rabbits and human, were examined. Results: In a mouse model of toxocariasis, intraperitoneal injection of T. canis larvae induces inflammatory leukocyte accumulation in the liver and the lungs but not in the brain, although a remarkable number of larvae were detected in this organ. Mice and rabbits responded differently to Toxocara spp. resulting in distinct heterogenous WB band patterns. Mice and rabbits both responded to a 33.1 kDa E/S constituent that turned out to be the most sensitive protein for serodiagnosis. Sera from human toxocariasis patients showed heterogenous WB band patterns similar to those observed in rabbits and all responded to the 33.1 kDa band. Conclusion: 33.1 kDa E/S protein can be considered as a critical common biomarker for toxocariasis immuno-diagnosis in both paratenic animals and human and its specificity requires further investigation. PMID:28761463

E1-Like Activating Enzyme Atg7 Is Preferentially Sequestered into p62 Aggregates via Its Interaction with LC3-I

PubMed Central

Gao, Wentao; Chen, Zhixia; Wang, Wei; Stang, Michael T.

2013-01-01

p62 is constitutively degraded by autophagy via its interaction with LC3. However, the interaction of p62 with LC3 species in the context of the LC3 lipidation process is not specified. Further, the p62-mediated protein aggregation’s effect on autophagy is unclear. We systemically analyzed the interactions of p62 with all known Atg proteins involved in LC3 lipidation. We find that p62 does not interact with LC3 at the stages when it is being processed by Atg4B or when it is complexed or conjugated with Atg3. p62 does interact with LC3-I and LC3-I:Atg7 complex and is preferentially recruited by LC3-II species under autophagic stimulation. Given that Atg4B, Atg3 and LC3-Atg3 are indispensable for LC3-II conversion, our study reveals a protective mechanism for Atg4B, Atg3 and LC3-Atg3 conjugate from being inappropriately sequestered into p62 aggregates. Our findings imply that p62 could potentially impair autophagy by negatively affecting LC3 lipidation and contribute to the development of protein aggregate diseases. PMID:24023838

E1-like activating enzyme Atg7 is preferentially sequestered into p62 aggregates via its interaction with LC3-I.

PubMed

Gao, Wentao; Chen, Zhixia; Wang, Wei; Stang, Michael T

2013-01-01

p62 is constitutively degraded by autophagy via its interaction with LC3. However, the interaction of p62 with LC3 species in the context of the LC3 lipidation process is not specified. Further, the p62-mediated protein aggregation's effect on autophagy is unclear. We systemically analyzed the interactions of p62 with all known Atg proteins involved in LC3 lipidation. We find that p62 does not interact with LC3 at the stages when it is being processed by Atg4B or when it is complexed or conjugated with Atg3. p62 does interact with LC3-I and LC3-I:Atg7 complex and is preferentially recruited by LC3-II species under autophagic stimulation. Given that Atg4B, Atg3 and LC3-Atg3 are indispensable for LC3-II conversion, our study reveals a protective mechanism for Atg4B, Atg3 and LC3-Atg3 conjugate from being inappropriately sequestered into p62 aggregates. Our findings imply that p62 could potentially impair autophagy by negatively affecting LC3 lipidation and contribute to the development of protein aggregate diseases.

Role of aerosil dispersion on the activated kinetics of the LC1-xSilx system.

PubMed

Sharma, Dipti; MacDonald, John C; Iannacchione, Germano S

2006-12-28

This study explores the role of aerosil dispersion on activated phase transitions of bulk octylcyanobiphenyl (8CB) liquid crystals by performing heating rate-dependent experiments. Differential scanning calorimetry (DSC) was used at various heating ramp rates in order to probe the activated phase dynamics of the system. The system, LC1-xSilx, was prepared by mixing aerosil nanoparticles (7 nm in diameter) in the bulk 8CB by the solvent dispersion method (SDM). LC represents bulk 8CB, and Sil represents aerosil nanoparticles with concentration x in percent. The concentration of the aerosil nanoparticles (x) varied from 0 to 0.2 g/cm3 in the bulk 8CB. Well-defined, endothermic peaks were found on a heating scan at melting and at the smectic-A to nematic (SmA-N) and nematic to isotropic (N-I) transitions. These peaks show a temperature shift and a change in their shapes and sizes in the presence of aerosil nanoparticles. In addition, an exothermic peak also appeared before the melting peak during the heating scan in the presence of aerosil nanoparticles. All transitions shifted significantly with different heating ramp rates, following an Arrhenius behavior, showing activated kinetics. The presence of aerosil nanoparticles caused a significant increase in the enthalpy and a decrease in the activation energy compared to the results found in bulk 8CB. This behavior can be explained by aerosil dispersion in the LC1-xSilx, inducing a disorder in the bulk 8CB. Infrared (IR) spectroscopy shows a shift to higher frequency for the broad peak at 1082 cm-1, corresponding to an Si-O bond as the density of the aerosil increases, and can be explained in terms of surface and molecular interactions between aerosil nanoparticles and 8CB liquid crystal molecules.

Identification of a major 50-kDa molecular weight human B-cell growth factor with Tac antigen-inducing activity on B cells.

PubMed

Kawano, M; Matsushima, K; Oppenheim, J J

1987-08-01

A bioassay was developed using human small B cells adherent to anti-human IgM (anti-mu)-coated wells. These B cells were stimulated to proliferate by culture supernatants of concanavalin A (Con A)-activated human peripheral blood lymphocytes (Con A Sup) even in the presence of high concentrations of anti-mu coated on assay wells. Human B-cell growth factor (BCGF) activities were partially purified from Con A Sup. Preparative chromatography (Sephacryl S-200 and isoelectrofocusing) yielded a major peak of BCGF activity for B cells adherent to anti-mu-coated wells with a molecular weight of 50,000 (50 kDa) and a pI 7.6. The 50-kDa BCGF was further purified by sequential chromatography using DEAE-Sephacel, CM-Sepharose, Sephacryl S-200, CM-high performance liquid chromatography (HPLC), and hydroxyapatite (HA)-HPLC. The HA-HPLC-purified 50-kDa BCGF was free of interleukin-1 (IL-1), interleukin-2 (IL-2), and interferon activities, but could support growth of BCL1 cells, similar to BCGF-II. Neither IL-1 nor interferon-gamma had any growth-stimulating effect in our B-cell proliferation assay with or without BCGF in Iscove's synthetic assay medium. BCGF-induced proliferation of B cells adherent to anti-mu-coated wells could be markedly augmented by the simultaneous or sequential addition of recombinant human IL-2 (rIL-2). When cultured for 3 days with 50-kDa BCGF, about 40% of B cells adherent to anti-mu-coated wells expressed Tac antigen, and monoclonal anti-Tac antibody inhibited rIL-2 enhancement of proliferation of 50-kDa BCGF-preactivated B cells. In addition, 50-kDa BCGF could induce Tac antigen on an Epstein-Barr virus-transformed B-cell line (ORSON) in the presence of a suboptimal dose of phorbol myristate acetate (PMA) and also on a natural killer-like cell line (YT cells). We have therefore identified a major 50-kDa BCGF activity with Tac antigen-inducing activity that also has a synergistic effect with IL-2 on normal B-cell proliferation.

Expression of autophagy related genes mTOR, Becline-1, LC3 and p62 in the peripheral blood mononuclear cells of systemic lupus erythematosus.

PubMed

Wu, Zhen-Zhen; Zhang, Jun-Jun; Gao, Cong-Cong; Zhao, Man; Liu, Sheng-Yun; Gao, Guan-Min; Zheng, Zhao-Hui

2017-01-01

To determine the expression of mTOR, Becline-1, LC3 and p62 in the peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus (SLE) and assess their relationship with disease activity and immunologic features. The expression of mTOR, Becline-1, LC3 and p62 was detected by RT-PCR in 81 SLE subjects and 86 age- and sex-matched healthy controls. Data regarding demographics and clinical parameters were collected. Disease activity of SLE was evaluated according to the SLE Disease Activity Index (SLEDAI) score. Independent sample t-test was used to analyze the expression of mTOR, Becline-1, LC3, and p62 in the two groups. Pearson's or Spearman's correlation was performed to analyze their relationship with disease activity and immunologic features. The mean levels of Becline-1, LC3 and p62 mRNA were significantly higher in SLE patients than the controls (9.96×10 -4 vs 7.38×10 -4 for Becline-1 with P <0.001; 4.04×10 -5 vs 2.62×10 -5 for LC3 with P <0.001; 9.51×10 -4 vs 7.59×10 -4 for p62 with P =0.008). However, the levels of mTOR mRNA in SLE patients were not significantly different from that in controls. Correlation analysis showed that Becline-1, LC3 and p62 mRNA levels correlated positively with SLEDAI, IgG and ds-DNA, negatively with C3. Our results suggested that autophagosomes formation were activated and their degradation were blocked in SLE. Moreover, the maintenance of autophagy balance can improve disease activity and immune disorders in SLE patients.

The influence of an external electric field on the propagation of light waves in cholesteric liquid crystal cells

NASA Astrophysics Data System (ADS)

Aksenova, E. V.; Karetnikov, A. A.; Kovshik, A. P.; Krainyukov, E. S.; Svanidze, A. V.

2017-05-01

The specific features of light transmission in a cholesteric liquid crystal (LC) cell with a director rotated by 90° have been investigated. In this structure, where a light wave is incident at a large angle with respect to the LC surface, the light is reflected (refracted) in the LC layer near the opposite boundary. It is shown that the application of an electric field changes the character of extraordinary wave refraction, as a result of which light starts passing through a cell. The transmission threshold voltage is determined, and its dependence on the angle of incidence of light is obtained. The dependence of the transmitted-light intensity on the voltage across the cell is obtained as well. The same dependences are also derived by numerical calculations with allowance for the turning points and extinction.

4D Light Field Imaging System Using Programmable Aperture

NASA Technical Reports Server (NTRS)

Bae, Youngsam

2012-01-01

Complete depth information can be extracted from analyzing all angles of light rays emanated from a source. However, this angular information is lost in a typical 2D imaging system. In order to record this information, a standard stereo imaging system uses two cameras to obtain information from two view angles. Sometimes, more cameras are used to obtain information from more angles. However, a 4D light field imaging technique can achieve this multiple-camera effect through a single-lens camera. Two methods are available for this: one using a microlens array, and the other using a moving aperture. The moving-aperture method can obtain more complete stereo information. The existing literature suggests a modified liquid crystal panel [LC (liquid crystal) panel, similar to ones commonly used in the display industry] to achieve a moving aperture. However, LC panels cannot withstand harsh environments and are not qualified for spaceflight. In this regard, different hardware is proposed for the moving aperture. A digital micromirror device (DMD) will replace the liquid crystal. This will be qualified for harsh environments for the 4D light field imaging. This will enable an imager to record near-complete stereo information. The approach to building a proof-ofconcept is using existing, or slightly modified, off-the-shelf components. An SLR (single-lens reflex) lens system, which typically has a large aperture for fast imaging, will be modified. The lens system will be arranged so that DMD can be integrated. The shape of aperture will be programmed for single-viewpoint imaging, multiple-viewpoint imaging, and coded aperture imaging. The novelty lies in using a DMD instead of a LC panel to move the apertures for 4D light field imaging. The DMD uses reflecting mirrors, so any light transmission lost (which would be expected from the LC panel) will be minimal. Also, the MEMS-based DMD can withstand higher temperature and pressure fluctuation than a LC panel can. Robotics need

Exploring on the Sensitivity Changes of the LC Resonance Magnetic Sensors Affected by Superposed Ringing Signals.

PubMed

Lin, Tingting; Zhou, Kun; Yu, Sijia; Wang, Pengfei; Wan, Ling; Zhao, Jing

2018-04-25

LC resonance magnetic sensors are widely used in low-field nuclear magnetic resonance (LF-NMR) and surface nuclear magnetic resonance (SNMR) due to their high sensitivity, low cost and simple design. In magnetically shielded rooms, LC resonance magnetic sensors can exhibit sensitivities at the fT/√Hz level in the kHz range. However, since the equivalent magnetic field noise of this type of sensor is greatly affected by the environment, weak signals are often submerged in practical applications, resulting in relatively low signal-to-noise ratios (SNRs). To determine why noise increases in unshielded environments, we analysed the noise levels of an LC resonance magnetic sensor ( L ≠ 0) and a Hall sensor ( L ≈ 0) in different environments. The experiments and simulations indicated that the superposed ringing of the LC resonance magnetic sensors led to the observed increase in white noise level caused by environmental interference. Nevertheless, ringing is an inherent characteristic of LC resonance magnetic sensors. It cannot be eliminated when environmental interference exists. In response to this problem, we proposed a method that uses matching resistors with various values to adjust the quality factor Q of the LC resonance magnetic sensor in different measurement environments to obtain the best sensitivity. The LF-NMR experiment in the laboratory showed that the SNR is improved significantly when the LC resonance magnetic sensor with the best sensitivity is selected for signal acquisition in the light of the test environment. (When the matching resistance is 10 kΩ, the SNR is 3.46 times that of 510 Ω). This study improves LC resonance magnetic sensors for nuclear magnetic resonance (NMR) detection in a variety of environments.

Light-melt adhesive based on dynamic carbon frameworks in a columnar liquid-crystal phase

NASA Astrophysics Data System (ADS)

Saito, Shohei; Nobusue, Shunpei; Tsuzaka, Eri; Yuan, Chunxue; Mori, Chigusa; Hara, Mitsuo; Seki, Takahiro; Camacho, Cristopher; Irle, Stephan; Yamaguchi, Shigehiro

2016-07-01

Liquid crystal (LC) provides a suitable platform to exploit structural motions of molecules in a condensed phase. Amplification of the structural changes enables a variety of technologies not only in LC displays but also in other applications. Until very recently, however, a practical use of LCs for removable adhesives has not been explored, although a spontaneous disorganization of LC materials can be easily triggered by light-induced isomerization of photoactive components. The difficulty of such application derives from the requirements for simultaneous implementation of sufficient bonding strength and its rapid disappearance by photoirradiation. Here we report a dynamic molecular LC material that meets these requirements. Columnar-stacked V-shaped carbon frameworks display sufficient bonding strength even during heating conditions, while its bonding ability is immediately lost by a light-induced self-melting function. The light-melt adhesive is reusable and its fluorescence colour reversibly changes during the cycle, visualizing the bonding/nonbonding phases of the adhesive.

Localization of Beclin1 in mouse developing tooth germs: possible implication of the interrelation between autophagy and apoptosis.

PubMed

Yang, Jingwen; Wan, Chunyan; Nie, Shuai; Jian, Shujuan; Sun, Zheyi; Zhang, Lu; Chen, Zhi

2013-12-01

Our previous study identified the appearance of autophagy in developing tooth germs, and suggested its possible association with apoptosis in odontogenesis. Beclin1 was recently indicated to play a central role in bridging autophagy and apoptosis, and occupied a key position in the process of development. This study hypothesized that Beclin1 may be involved, and act as the molecular basis of the connection between autophagy and apoptosis in odontogenesis. Immunohistochemical analysis showed the spatiotemporal expression pattern of Beclin1 in odontogenesis from embryonic (E) day 13.5 to postnatal (P) day 5.5. At E stages, Beclin1 was mainly immunolocalized in the cytoplasm of the cells in the enamel organ. Meanwhile, the nucleus localization of Beclin1 was detected in part of the stellate reticulum, outer and inner enamel epithelium, especially at E16.5 and E18.5. At P stages, Beclin1 was detected in the cytoplasm of the odontoblasts, besides the dental epithelium cells. Triple immunofluorescence analysis showed the partial colocalization of Beclin1, autophagic marker LC3, or activated caspase-3 in the E14.5 tooth germs, especially the Beclin1(+)LC3(+)Caspase-3(+) cells in the PEK. Furthermore, western blot analysis revealed that the full-length (60 kDa) and/or cleaved (50, 37, and 35 kDa) Beclin1 in the developing tooth germs. Taken together, our findings indicate that Beclin1 is involved, and might be responsible for the crosstalk between autophagy and apoptosis in mouse odontogenesis.

Differential recruitment efficacy of patient-derived amyloidogenic and myeloma light chain proteins by synthetic fibrils-A metric for predicting amyloid propensity.

PubMed

Martin, Emily B; Williams, Angela; Wooliver, Craig; Heidel, R Eric; Adams, Sarah; Dunlap, John; Ramirez-Alvarado, Marina; Blancas-Mejia, Luis M; Lands, Ronald H; Kennel, Stephen J; Wall, Jonathan S

2017-01-01

Monoclonal free light chain (LC) proteins are present in the circulation of patients with immunoproliferative disorders such as light chain (AL) amyloidosis and multiple myeloma (MM). Light chain-associated amyloid is a complex pathology composed of proteinaceous fibrils and extracellular matrix proteins found in all patients with AL and in ~10-30% of patients who presented with MM. Amyloid deposits systemically in multiple organs and tissues leading to dysfunction and ultimately death. The overall survival of patients with amyloidosis is worse than for those with early stage MM. We have developed a sensitive binding assay quantifying the recruitment of full length, patient-derived LC proteins by synthetic amyloid fibrils, as a method for studying their amyloidogenic potential. In a survey of eight urinary LC, both AL and MM-associated proteins were recruited by synthetic amyloid fibrils; however, AL-associated LC bound significantly more efficiently (p < 0.05) than did MM LCs. The LC proteins used in this study were isolated from urine and presumed to represent a surrogate of serum free light chains. The binding of LC to synthetic fibrils in this assay accurately differentiated LC with amyloidogenic propensity from MM LC that were not associated with clinical amyloid disease. Notably, the LC from a MM patient who subsequently developed amyloid behaved as an AL-associated protein in the assay, indicating the possibility for identifying MM patients at risk for developing amyloidosis based on the light chain recruitment efficacy. With this information, at risk patients can be monitored more closely for the development of amyloidosis, allowing timely administration of novel, amyloid-directed immunotherapies-this approach may improve the prognosis for these patients.

Methods for the analysis of organophosphorus flame retardants-Comparison of GC-EI-MS, GC-NCI-MS, LC-ESI-MS/MS, and LC-APCI-MS/MS.

PubMed

Tokumura, Masahiro; Miyake, Yuichi; Wang, Qi; Nakayama, Hayato; Amagai, Takashi; Ogo, Sayaka; Kume, Kazunari; Kobayashi, Takeshi; Takasu, Shinji; Ogawa, Kumiko

2018-04-16

Organophosphorus flame retardants (PFRs) are extensively used as alternatives to banned polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD). In this study, we analyzed 14 PFRs by means of four mass-spectrometry-based methods: gas chromatography combined with electron-impact mass spectrometry (GC-EI-MS) or negative-chemical-ionization mass spectrometry (GC-NCI-MS) and liquid chromatography combined with tandem mass spectrometry using electrospray ionization (LC-ESI-MS/MS) or atmospheric pressure chemical ionization (LC-APCI-MS/MS). The limits of quantification (LOQs) for LC-ESI-MS/MS and LC-APCI-MS/MS (0.81-970 pg) were 1-2 orders of magnitude lower than the LOQs for GC-EI-MS and GC-NCI-MS (2.3-3900 pg). LC-APCI-MS/MS showed the lowest LOQs (mean = 41 pg; median = 3.4 pg) for all but two of the PFRs targeted in this study. For LC-APCI-MS/MS, the lowest LOQ was observed for tributyl phosphate (TBP) (0.81 pg), and the highest was observed for tris(butoxyethyl) phosphate (TBOEP) (36 pg). The results of this study indicate that LC-APCI-MS/MS is the optimum analytical method for the target PFRs, at least in terms of LOQ.

Red light-induced formation of ubiquitin-phytochrome conjugates: Identification of possible intermediates of phytochrome degradation

PubMed Central

Shanklin, John; Jabben, Merten; Vierstra, Richard D.

1987-01-01

Phytochrome is the photoreceptor that controls red light-mediated morphogenesis in higher plants. It exists in two photointerconvertible forms, a red light-absorbing form, Pr, and a far-red light-absorbing form, Pfr. Because photoconversion of Pr to Pfr by a brief light pulse decreases the in vivo half-life of this chromoprotein by a factor of ≈100, this system offers a unique way to modulate the turnover rate of a specific protein and hence study the mechanisms responsible for selective protein degradation. In etiolated oat [Avena sativa (L.)] seedlings, degradation of phytochrome as Pfr follows zero-order kinetics as measured both spectrally and by ELISA, with 50% of Pfr lost in ≈130 min at 27°C. Immunoblot analysis of the destruction process with anti-oat phytochrome immunoglobulins reveals that degradation involves the loss of the 124-kDa phytochrome monomer and that proteolytic intermediates of apparent molecular mass lower than 124 kDa do not accumulate to detectable levels in vivo (<0.015% of total phytochrome). The latter observation suggests that proteolytic breakdown of the protein is extremely rapid. However, a series of polypeptides with higher apparent molecular mass and recognized by anti-phytochrome immunoglobulins (principally 129 and 134 kDa) appears after photoconversion to Pfr. These polypeptides represent no more than a few percent of the total immunologically detectable phytochrome pool and have incremental differences in apparent molecular mass of 5 kDa. They appear within 5 min after Pfr formation, reach maximal levels between 90 and 180 min, and decline thereafter. These polypeptides and others of apparent molecular mass up to 160 kDa are also detectable with immunoglobulins directed against either oat or human ubiquitin, indicating that they are ubiquitin-phytochrome conjugates. Since ubiquitin conjugation is involved in intracellular protein turnover and since formation and degradation of Pfr-ubiquitin conjugates coincide with the

The C terminus of the catalytic domain of type A botulinum neurotoxin may facilitate product release from the active site.

PubMed

Mizanur, Rahman M; Frasca, Verna; Swaminathan, Subramanyam; Bavari, Sina; Webb, Robert; Smith, Leonard A; Ahmed, S Ashraf

2013-08-16

Botulinum neurotoxins are the most toxic of all compounds. The toxicity is related to a poor zinc endopeptidase activity located in a 50-kDa domain known as light chain (Lc) of the toxin. The C-terminal tail of Lc is not visible in any of the currently available x-ray structures, and it has no known function but undergoes autocatalytic truncations during purification and storage. By synthesizing C-terminal peptides of various lengths, in this study, we have shown that these peptides competitively inhibit the normal catalytic activity of Lc of serotype A (LcA) and have defined the length of the mature LcA to consist of the first 444 residues. Two catalytically inactive mutants also inhibited LcA activity. Our results suggested that the C terminus of LcA might interact at or near its own active site. By using synthetic C-terminal peptides from LcB, LcC1, LcD, LcE, and LcF and their respective substrate peptides, we have shown that the inhibition of activity is specific only for LcA. Although a potent inhibitor with a Ki of 4.5 μm, the largest of our LcA C-terminal peptides stimulated LcA activity when added at near-stoichiometric concentration to three versions of LcA differing in their C-terminal lengths. The result suggested a product removal role of the LcA C terminus. This suggestion is supported by a weak but specific interaction determined by isothermal titration calorimetry between an LcA C-terminal peptide and N-terminal product from a peptide substrate of LcA. Our results also underscore the importance of using a mature LcA as an inhibitor screening target.

On-Line 1D and 2D PLOT/LC-ESI-MS Using 10 μm i.d. Poly(styrene–divinylbenzene) Porous Layer Open Tubular (PLOT) Columns For Ultrasensitive Proteomic Analysis

PubMed Central

Luo, Quanzhou; Yue, Guihua; Valaskovic, Gary A; Gu, Ye; Wu, Shiaw-Lin; Karger, Barry L.

2008-01-01

Following on our recent work, on-line one dimensional (1D) and two dimensional (2D) PLOT/LC-ESI-MS platforms using 3.2 m × 10 μm i.d. poly(styrenedivinylbenzene) (PS-DVB) porous layer open tubular (PLOT) columns have been developed to provide robust, high performance and ultrasensitive proteomic analysis. Using a PicoClear tee, the dead volume connection between a 50 μm i.d. PS-DVB monolithic microSPE column and the PLOT column was minimized. The microSPE/PLOT column assembly provided a separation performance similar to that obtained with direct injection onto the PLOT column at a mobile phase flow rate of 20 nL/min. The trace analysis potential of the platform was evaluated using an in-gel tryptic digest sample of a gel fraction (15 to 40 kDa) of a cervical cancer (SiHa) cell line. As an example of the sensitivity of the system, ∼2.5 ng of protein in 2 μL solution, an amount corresponding to 20 SiHa cells, was subjected to on-line microSPE-PLOT/LC-ESIMS/MS analysis using a linear ion trap MS. 237 peptides associated with 163 unique proteins were identified from a single analysis when using stringent criteria associated with a false positive rate less than 1% . The number of identified peptides and proteins increased to 638 and 343, respectively, as the injection amount was raised to ∼45 ng of protein, an amount corresponding to 350 SiHa cells. In comparison, only 338 peptides and 231 unique proteins were identified (false positive rate again less than 1%) from 750 ng of protein from the identical gel fraction, an amount corresponding to 6000 SiHa cells, using a typical 15 cm × 75 μm i.d. packed capillary column. The greater sensitivity, higher recovery, and higher resolving power of the PLOT column resulted in the increased number of identifications from only ∼5% of the injected sample amount. The resolving power of the microSPE/PLOT assembly was further extended by 2D chromatography via combination of the high-efficiency reversed phase PLOT column

The comparison of CHCA solvent compositions for improving LC-MALDI performance and its application to study the impact of aflatoxin B1 on the liver proteome of diabetes mellitus type 1 mice.

PubMed

Tsai, Fuu-Jen; Chen, Shih-Yin; Liu, Yu-Ching; Liao, Hsin-Yi; Chen, Chao-Jung

2017-01-01

In nanoflow liquid chromatography-matrix-assisted laser desorption/ionization tandem time-of-flight (nanoLC-MALDI-TOF/TOF) approaches, it is critical to directly apply small amounts of the sample elutes on the sample target using a nanoLC system due to its low flow rate of 200 ~ 300 nl/min. It is recommended to apply a sheath liquid containing a matrix with a several μL/min flow rate at the end of the nanoLC column to ensure a larger co-eluted droplet for more reproducible sample spotting and avoid the laborious task of post-manual matrix spotting. In this study, to achieve a better nanoLC-MALDI performance on sample spotting, we first compared α-Cyano-4-hydroxycinnamic acid (CHCA) solvent composition for efficiently concentrating nanoLC elutes on an anchor chip. The solvent composition of isopropanol (IPA): acetonitrile (ACN):acetone:0.1% Trifluoroacetic acid (TFA) (2:7:7:2) provided strong and homogeneous signals with higher peptide ion yields than the other solvent compositions. Then, nanoLC-MALDI-TOF/TOF was applied to study the impact of aflatoxin B1 on the liver proteome from diabetes mellitus type 1 mice. Aflatoxin B1 (AFB1), produced by Aspergillus flavus and Aspergillus parasiticus is a carcinogen and a known causative agent of liver cancer. To evaluate the effects of long-term exposure to AFB1 on type 1 diabetes mellitus (TIDM), the livers of T1DM control mice and mice treated with AFB1 were analyzed using isotope-coded protein labeling (ICPL)-based quantitative proteomics. Our results showed that gluconeogenesis, lipid, and oxidative phosphorylation mechanisms, normally elevated in T1DM, were disordered following AFB1 treatment. In addition, major urinary protein 1 (MUP1), an indicator of increased insulin sensitivity, was significantly decreased in the T1DM/AFB1 group and may have resulted in higher blood glucose levels compared to the T1DM group. These results indicate that T1DM patients should avoid the AFB1 intake, as they could lead to increased

Solution Structure of the 128 kDa Enzyme I Dimer from Escherichia coli and Its 146 kDa Complex with HPr Using Residual Dipolar Couplings and Small- and Wide-Angle X-ray Scattering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwieters, Charles D.; Suh, Jeong-Yong; Grishaev, Alexander

2010-09-17

The solution structures of free Enzyme I (EI, {approx}128 kDa, 575 x 2 residues), the first enzyme in the bacterial phosphotransferase system, and its complex with HPr ({approx}146 kDa) have been solved using novel methodology that makes use of prior structural knowledge (namely, the structures of the dimeric EIC domain and the isolated EIN domain both free and complexed to HPr), combined with residual dipolar coupling (RDC), small- (SAXS) and wide- (WAXS) angle X-ray scattering and small-angle neutron scattering (SANS) data. The calculational strategy employs conjoined rigid body/torsion/Cartesian simulated annealing, and incorporates improvements in calculating and refining against SAXS/WAXS datamore » that take into account complex molecular shapes in the description of the solvent layer resulting in a better representation of the SAXS/WAXS data. The RDC data orient the symmetrically related EIN domains relative to the C{sub 2} symmetry axis of the EIC dimer, while translational, shape, and size information is provided by SAXS/WAXS. The resulting structures are independently validated by SANS. Comparison of the structures of the free EI and the EI-HPr complex with that of the crystal structure of a trapped phosphorylated EI intermediate reveals large ({approx}70-90{sup o}) hinge body rotations of the two subdomains comprising the EIN domain, as well as of the EIN domain relative to the dimeric EIC domain. These large-scale interdomain motions shed light on the structural transitions that accompany the catalytic cycle of EI.« less

Analysis of the low molecular weight fraction of serum by LC-dual ESI-FT-ICR mass spectrometry: precision of retention time, mass, and ion abundance.

PubMed

Johnson, Kenneth L; Mason, Christopher J; Muddiman, David C; Eckel, Jeanette E

2004-09-01

This study quantifies the experimental uncertainty for LC retention time, mass measurement precision, and ion abundance obtained from replicate nLC-dual ESI-FT-ICR analyses of the low molecular weight fraction of serum. We used ultrafiltration to enrich the < 10-kDa fraction of components from the high-abundance proteins in a pooled serum sample derived from ovarian cancer patients. The THRASH algorithm for isotope cluster detection was applied to five replicate nLC-dual ESI-FT-ICR chromatograms. A simple two-level grouping algorithm was applied to the more than 7000 isotope clusters found in each replicate and identified 497 molecular species that appeared in at least four of the replicates. In addition, a representative set of 231 isotope clusters, corresponding to 188 unique molecular species, were manually interpreted to verify the automated algorithm and to set its tolerances. For nLC retention time reproducibility, 95% of the 497 species had a 95% confidence interval of the mean of +/- 0.9 min or less without the use of chromatographic alignment procedures. Furthermore, 95% of the 497 species had a mass measurement precision of < or = 3.2 and < or = 6.3 ppm for internally and externally calibrated spectra, respectively. Moreover, 95% of replicate ion abundance measurements, covering an ion abundance range of approximately 3 orders of magnitude, had a coefficient of variation of less than 62% without using any normalization functions. The variability of ion abundance was independent of LC retention time, mass, and ion abundance quartile. These measures of analytical reproducibility establish a statistical rationale for differentiating healthy and disease patient populations for the elucidation of biomarkers in the low molecular fraction of serum. Copyright 2004 American Chemical Society

DYNLL/LC8 Protein Controls Signal Transduction through the Nek9/Nek6 Signaling Module by Regulating Nek6 Binding to Nek9

PubMed Central

Regué, Laura; Sdelci, Sara; Bertran, M. Teresa; Caelles, Carme; Reverter, David; Roig, Joan

2011-01-01

The NIMA family protein kinases Nek9/Nercc1 and the highly similar Nek6 and Nek7 form a signaling module activated in mitosis, when they are involved in the control of spindle organization and function. Here we report that Nek9, the module upstream kinase, binds to DYNLL/LC8, a highly conserved protein originally described as a component of the dynein complex. LC8 is a dimer that interacts with different proteins and has been suggested to act as a dimerization hub promoting the organization and oligomerization of partially disorganized partners. We find that the interaction of LC8 with Nek9 depends on a (K/R)XTQT motif adjacent to the Nek9 C-terminal coiled coil motif, results in Nek9 multimerization, and increases the rate of Nek9 autoactivation. LC8 binding to Nek9 is regulated by Nek9 activity through the autophosphorylation of Ser944, a residue immediately N-terminal to the (K/R)XTQT motif. Remarkably, LC8 binding interferes with the interaction of Nek9 with its downstream partner Nek6 as well as with Nek6 activation, thus controlling both processes. Our work sheds light into the control of signal transduction through the module formed by Nek9 and Nek6/7 and uncovers a novel manner in which LC8 can regulate partner physiology by interfering with protein complex formation. We suggest that this and other LC8 functions can be specifically regulated by partner phosphorylation. PMID:21454704

A conserved 19-kDa Eimeria tenella antigen is a profilin-like protein.

PubMed

Fetterer, R H; Miska, K B; Jenkins, M C; Barfield, R C

2004-12-01

A wide range of recombinant proteins from Eimeria species have been reported to offer some degree of protection against infection and disease, but the specific biological function of these proteins is largely unknown. Previous studies have demonstrated a 19-kDa protein of unknown function designated SZ-1 in sporozoites and merozoites of Eimeria acervulina that can be used to confer partial protection against coccidiosis. Reverse transcriptase-polymerase chain reaction indicated that the gene for SZ-1 is expressed by all the asexual stages of Eimeria tenella. Rabbit antisera to recombinant SZ-1 recognized an approximately 19-kDa protein from extracts of E. tenella sporozoites, merozoites, sporulated oocysts, and oocysts in various stages of sporulation. Immunofluorescence antibody staining indicated specific staining of E. tenella sporozoites and merozoites. Staining was most intense in the cytoplasm of the posterior end of the parasite. The primary amino acid sequence of the gene for E. tenella SZ-1 deduced from the E. tenella genome indicated a conserved domain for the actin-regulatory protein profilin. A conserved binding site for poly-L-proline (PLP), characteristic of profilin was also observed. SZ-1 was separated from soluble extract of E. tenella proteins by affinity chromatography using a PLP ligand, confirming the ability of SZ-1 to bind PLP. SZ-1 also partially inhibited the polymerization of actin. The current results are consistent with the classification of SZ-1 as a profilin-related protein.

Rho-associated kinase plays a role in rabbit urethral smooth muscle contraction, but not via enhanced myosin light chain phosphorylation.

PubMed

Walsh, Michael P; Thornbury, Keith; Cole, William C; Sergeant, Gerard; Hollywood, Mark; McHale, Noel

2011-01-01

The involvement of Rho-associated kinase (ROK) in activation of rabbit urethral smooth muscle contraction was investigated by examining the effects of two structurally distinct inhibitors of ROK, Y27632 and H1152, on the contractile response to electric field stimulation, membrane depolarization with KCl, and α1-adrenoceptor stimulation with phenylephrine. Both compounds inhibited contractions elicited by all three stimuli. The protein kinase C inhibitor GF109203X, on the other hand, had no effect. Urethral smooth muscle strips were analyzed for phosphorylation of three potential direct or indirect substrates of ROK: 1) myosin regulatory light chains (LC20) at S19, 2) the myosin-targeting subunit of myosin light chain phosphatase (MYPT1) at T697 and T855, and 3) cofilin at S3. The following results were obtained: 1) under resting tension, LC20 was phosphorylated to 0.65±0.02 mol Pi/mol LC20 (n=21) at S19; 2) LC20 phosphorylation did not change in response to KCl or phenylephrine; 3) ROK inhibition had no effect on LC20 phosphorylation in the absence or presence of contractile stimuli; 4) under resting conditions, MYPT1 was partially phosphorylated at T697 and T855 and cofilin at S3; 5) phosphorylation of MYPT1 and cofilin was unaffected by KCl or phenylephrine; and 6) KCl- and phenylephrine-induced contraction-relaxation cycles did not correlate with actin polymerization-depolymerization. We conclude that ROK plays an important role in urethral smooth muscle contraction, but not via inhibition of MLCP or polymerization of actin.

12. INTERIOR OF KITCHEN/UTILITY AREA SHOWING ORIGINAL 1LIGHT OVER 1LIGHT, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

12. INTERIOR OF KITCHEN/UTILITY AREA SHOWING ORIGINAL 1-LIGHT OVER 1-LIGHT, DOUBLE HUNG WINDOW OVER SINK, AND BUILT-IN KITCHEN CABINETRY. VIEW TO SOUTHEAST. - Bishop Creek Hydroelectric System, Plant 4, Worker Cottage, Bishop Creek, Bishop, Inyo County, CA

High expression of 23 kDa protein of augmenter of liver regeneration (ALR) in human hepatocellular carcinoma

PubMed Central

Yu, Hai-Ying; Zhu, Man-Hua; Xiang, Dai-Rong; Li, Jun; Sheng, Ji-Fang

2014-01-01

Background Augmenter of liver regeneration (ALR) is an important polypeptide that participates in the process of liver regeneration. Two forms of ALR proteins are expressed in hepatocytes. Previous data have shown that ALR is essential for cell survival and has potential antimetastatic properties in hepatocellular carcinoma (HCC). Aims The study aimed to evaluate the expression levels of two forms of ALR proteins in HCC and their possible significance in HCC development. Methods Balb/c mouse monoclonal antibody against ALR protein was prepared in order to detect the ALR protein in HCC by Western blotting and immunohistochemistry. ALR mRNA expression levels were measured by real-time polymerase chain reaction in HCC tissues and compared to paracancerous liver tissues in 22 HCC patients. Results ALR mRNA expression in HCC liver tissues (1.51×106 copies/μL) was higher than in paracancerous tissues (1.04×104 copies/μL). ALR protein expression was also enhanced in HCC liver tissues. The enhanced ALR protein was shown to be 23 kDa by Western blotting. Immunohistochemical analysis showed that the 23 kDa ALR protein mainly existed in the hepatocyte cytosol. Conclusion The 23 kDa ALR protein was highly expressed in HCC and may play an important role in hepatocarcinogenesis. PMID:24940072

Optically addressed and submillisecond response phase only liquid crystal spatial light modulator

NASA Astrophysics Data System (ADS)

Zhao, Xiangjie; Duan, Jiazhu; Zhang, Dayong; Luo, Yongquan

2014-10-01

Liquid crystal based phase only spatial light modulator has attracted many research interests since last decades because of its superior advantage. Until now the liquid crystal spatial light modulator has been applied in many fields, but the response speed of nematic LC limited its further application. In this paper, an optically addressed phase only LC spatial light modulator was proposed based on polymer network liquid crystal. Morphology effect on the light scattering of PNLC was studied, which was mainly consisted of fiber and fiber bundles. The morphology nearly determined the light scattering and electro-optical property. Due to the high threshold voltage, to address the PNLC phase modulator was also concerned. Optical addressing method was proposed, in which BSO crystal was selected to replace one of the glass substrate. The response speed of PNLC was so fast that the reorientation of liquid crystal director will follow the change of effective voltage applied on LC layer, which was related with the voltage signal and especially with electron transport of photo-induced carriers due to diffusion and drift. The on state dynamic response of phase change was investigated. Based on this device, beam steering was also achieved by loading 488nm laser strip on the optical addressed phase only spatial light modulator.

Quantity and functionality of protein fractions in chicken breast fillets affected by white striping.

PubMed

Mudalal, S; Babini, E; Cavani, C; Petracci, M

2014-08-01

Recently, white striations parallel to muscle fibers direction have been observed on the surface of chicken breast, which could be ascribed to intensive growth selection. The aim of this study was to evaluate the effect of white striping on chemical composition with special emphasis on myofibrillar and sarcoplasmic protein fractions that are relevant to the processing features of chicken breast meat. During this study, a total of 12 pectoralis major muscles from both normal and white striped fillets were used to evaluate chemical composition, protein solubility (sarcoplasmic, myofibrillar, and total protein solubility), protein quantity (sarcoplasmic, myofibrillar, and stromal proteins), water holding capacity, and protein profile by SDS-PAGE analysis. White-striped fillets exhibited a higher percentage of moisture (75.4 vs. 73.8%; P < 0.01), intramuscular fat (2.15 vs. 0.98%; P < 0.01), and collagen (1.36 vs. 1.22%; P < 0.01), and lower content of protein (18.7 vs. 22.8%; P < 0.01) and ash (1.14 vs. 1.34%; P < 0.01), in comparison with normal fillets. There was a great decline in myofibrillar (14.0 vs. 8.7%; P < 0.01) and sarcoplasmic (3.2 vs. 2.6%; P < 0.01) content and solubility as well as an increase in cooking loss (33.7 vs. 27.4%; P < 0.05) due to white striping defects. Moreover, gel electrophoresis showed that the concentration of 3 myofibrillar proteins corresponding to actin (42 kDa); LC1, slow-twitch light chain myosin (27.5 kDa); and LC3, fast-twitch light chain myosin (16 kDa), and almost all sarcoplasmic proteins were lower than normal. In conclusion, the findings of this study revealed that chicken breast meat with white striping defect had different chemical composition (more fat and less protein) and protein quality and quantity (low content of myofibrillar proteins and high content of stromal proteins) with respect to normal meat. Furthermore, white striped fillets had lower protein functionality (higher cooking loss). All the former changes

Quantification of CSF cystatin C using liquid chromatography tandem mass spectrometry.

PubMed

Matsuda, Chikashi; Shiota, Yuri; Sheikh, Abdullah Md; Okazaki, Ryota; Yamada, Kazuo; Yano, Shozo; Minohata, Toshikazu; Matsumoto, Ken-Ichi; Yamaguchi, Shuhei; Nagai, Atsushi

2018-03-01

Cystatin C (CST3), a ubiquitously expressed cysteine protease inhibitor, is implicated in several neurological diseases. Here, we have developed an accurate CST3 measurement system based on liquid chromatography tandem mass spectrometry (LC-MS/MS). LC-MS/MS based measurement for CSF CST3 was validated by determination of assay precision, accuracy and recovery. The values were compared with those measured by immunoassay. Glycosylation of CST3 in CSF was analyzed by Western blotting and lectin blotting. Measuring standard CST3 by LC-MS/MS produced a linear standard curve that correlated with assigned values (r 2 =0.99). Both intra- and inter-assay variation was <10%. Although showed a correlation, the average CST3 concentration measured by LC-MS/MS was significantly higher than that of immunoassay. Western blotting showed the presence of a 25KDa species along with CST3 monomer (14KDa) in CSF. The volume of 25KDa species was decreased by deglycosylation. Lectin blotting revealed a 25KDa glycosylated protein in sialidase-treated CSF, which was decreased by deglycosylation. However, deglycosylation did not alter CST3 concentration measured by immunoassay. Our results suggest that LC-MS/MS-based CST3 measurement is a robust method with higher detection ability. Such method could be useful for the diagnosis and monitoring of neurological diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeting LC3 and Beclin-1 autophagy genes suppresses proliferation, survival, migration and invasion by inhibition of Cyclin-D1 and uPAR/Integrin β1/ Src signaling in triple negative breast cancer cells.

PubMed

Hamurcu, Zuhal; Delibaşı, Nesrin; Geçene, Seda; Şener, Elif Funda; Dönmez-Altuntaş, Hamiyet; Özkul, Yusuf; Canatan, Halit; Ozpolat, Bulent

2018-03-01

Autophagy is a catabolic process for degrading dysfunctional proteins and organelles, and closely associated with cancer cell survival under therapeutic, metabolic stress, hypoxia, starvation and lack of growth factors, contributing to resistance to therapies. However, the role of autophagy in breast cancer cells is not well understood. In the present study, we investigated the role of autophagy in highly aggressive and metastatic triple negative breast cancer (TNBC) and non-metastatic breast cancer cells and demonstrated that the knockdown of autophagy-related genes (LC3 and Beclin-1) inhibited autophagy and significantly suppressed cell proliferation, colony formation, migration/invasion and induced apoptosis in MDA-MB-231 and BT-549 TNBC cells. Knockdown of LC3 and Beclin-1 led to inhibition of multiple proto-oncogenic signaling pathways, including cyclin D1, uPAR/integrin-β1/Src, and PARP1. In conclusion, our study suggests that LC3 and Beclin-1 are required for cell proliferation, survival, migration and invasion, and may contribute to tumor growth and progression of highly aggressive and metastatic TNBC cells and therapeutic targeting of autophagy genes may be a potential therapeutic strategy for TNBC in breast cancer.

The characteristics of polyimide photoalignment layer with chalcone derivatives produced by linear polarized UV light

NASA Astrophysics Data System (ADS)

Jung, Kyoung Hoon; Hyun, Soon-Young; Song, Dong-Mee; Shin, Dong-Myung

2003-01-01

The photoalignment of liquid crystal (LC) molecules located onto polyimide films with chalcone derivatives using linearly polarized UV (LPUV) light is investigated. The LPUV light irradiation generated dimerization products of the chalcones followed by isomerization of the chalcone derivatives. The alignment directions of LC molecules were either homeotropic or planar with respect to plane of polyimide film, depending upon the alkyl chain length attached on the chalcones.

LC-MS and 1H NMR as an improved dereplication tool to identify antifungal diterpenoids from Sagittaria latifolia

USDA-ARS?s Scientific Manuscript database

A dereplication strategy using a combination of liquid chromatography-mass spectrometry (LC-MS) and proton nuclear magnetic resonance spectroscopy (1H NMR) to facilitate compound identification towards antifungal natural product discovery is presented. This analytical approach takes advantage of th...

Determination of Insecticidal Effect (LC50 and LC90) of Organic Fatty Acids Mixture (C8910+Silicone) Against Aedes aegypti and Aedes albopictus (Diptera: Culicidae).

PubMed

Dunford, James C; Falconer, Aneika; Leite, Laura N; Wirtz, Robert A; Brogdon, William G

2016-05-01

Emerging and re-emerging vector-borne diseases such as chikungunya and dengue and associated Aedes vectors are expanding their historical ranges; thus, there is a need for the development of novel insecticides for use in vector control programs. The mosquito toxicity of a novel insecticide and repellent consisting of medium-chain carbon fatty acids (C8910) was examined. Determination of LC 50 and LC 90 was made against colony-reared Aedes aegypti (L.) and Aedes albopictus (Skuse) using probit analysis on mortality data generated by Centers for Disease Control and Prevention bottle bioassays. Six different concentrations of C8910 + silicone oil yielded an LC 50 of 160.3 µg a.i/bottle (147.6-182.7) and LC 90 of 282.8 (233.2-394.2) in Ae. aegypti; five concentrations yielded an LC 50 of 125.4 (116.1-137.6) and LC 90 of 192.5 (165.0-278.9) in Ae. albopictus. Further development of C8910 and similar compounds could provide vector control specialists novel insecticides for controlling insect disease vectors. Published by Oxford University Press on behalf of Entomological Society of America 2015. This work is written by US Government employees and is in the public domain in the US.

Molecular characterization of a 40 kDa OmpC-like porin from Serratia marcescens.

PubMed

Hutsul, J A; Worobec, E

1994-02-01

An oligonucleotide that encodes the N-terminal portion of a 41 kDa porin of Serratia marcescens was used to probe S. marcescens UOC-51 genomic DNA. An 11 kb EcoRI fragment which hybridized with the oligonucleotide was subcloned into Escherichia coli, examined for expression, and sequenced. The product expressed by the cloned gene was 40 kDa. The nucleotide sequence has an ORF of 1.13 kb. When the deduced amino acid sequence was aligned and compared to other enterobacterial porins the cloned S. marcescens porin most closely resembled E. coli OmpC. Although we did not detect osmoregulation or thermoregulation of any porins in S. marcescens UOC-51, sequences analogous to the E. coli osmoregulator OmpR-binding regions are seen upstream to the cloned gene. We examined the regulation of the S. marcescens porin in E. coli and found that its expression increased in a high salt environment. A micF gene, whose transcriptional product functions to inhibit synthesis of OmpF by hybridizing with the ompF transcript, was also seen upstream of the S. marcescens ompC. An alignment with the E. coli micF gene revealed that the functional region of the S. marcescens micF gene is conserved. Based on the results obtained we have determined that S. marcescens UOC-51 produces a 40 kDa porin similar to the E. coli OmpC porin.

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

PubMed

Radford, N B; Fina, M; Benjamin, I J; Moreadith, R W; Graves, K H; Zhao, P; Gavva, S; Wiethoff, A; Sherry, A D; Malloy, C R; Williams, R S

1996-03-19

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.

Evaluation of peptide adsorption-controlled liquid chromatography-tandem mass spectrometric (PAC-LC-MS/MS) method for simple and simultaneous quantitation of amyloid β 1-38, 1-40, 1-42 and 1-43 peptides in dog cerebrospinal fluid.

PubMed

Goda, Ryoya; Kobayashi, Nobuhiro

2012-05-01

To evaluate the usefulness of the peptide adsorption-controlled liquid chromatography-tandem mass spectrometry (PAC-LC-MS/MS) for reproducible measurement of peptides in biological fluids, simultaneous quantitation of amyloid β 1-38, 1-40, 1-42 and 1-43 peptides (Aβ38, Aβ40, Aβ42 and Aβ43) in dog cerebrospinal fluid (CSF) was tried. Each stable isotope labeled Aβ was used as the internal standard to minimize the influence of CSF matrix on the reproducible Aβ quantitation. To reduce a loss of Aβ during the pretreatment procedures, the dog CSF diluted by water-acetic acid-methanol (2:6:1, v/v/v) was loaded on PAC-LC-MS/MS directly. Quantification of the Aβ in the diluted dog CSF was carried out using multiple reaction monitoring (MRM) mode. The [M+5H(5+)] and b(5+) ion fragment of each peptide were chosen as the precursor and product ions for MRM transitions of each peptide. The calibration curves were drawn from Aβ standard calibration solutions using PAC-LC-MS/MS. Analysis of dog CSF samples suggests that the basal concentration of Aβ38, Aβ40, Aβ42 and Aβ43 in dog CSF is approximately 300, 900, 200 and 30 pM, respectively. This is the first time Aβ concentrations in dog CSF have been reported. Additionally, the evaluation of intra- and inter-day reproducibility of analysis of Aβ standard solution, the freeze-thaw stability and the room temperature stability of Aβ standard solution suggest that the PAC-LC-MS/MS method enables reproducible Aβ quantitation. Copyright © 2012 Elsevier B.V. All rights reserved.

Host and Bacterial Proteins That Repress Recruitment of LC3 to Shigella Early during Infection

PubMed Central

Baxt, Leigh A.; Goldberg, Marcia B.

2014-01-01

Shigella spp. are intracytosolic gram-negative pathogens that cause disease by invasion and spread through the colonic mucosa, utilizing host cytoskeletal components to form propulsive actin tails. We have previously identified the host factor Toca-1 as being recruited to intracellular S. flexneri and being required for efficient bacterial actin tail formation. We show that at early times during infection (40 min.), the type three-secreted effector protein IcsB recruits Toca-1 to intracellular bacteria and that recruitment of Toca-1 is associated with repression of recruitment of LC3, as well as with repression of recruitment of the autophagy marker NDP52, around these intracellular bacteria. LC3 is best characterized as a marker of autophagosomes, but also marks phagosomal membranes in the process LC3-associated phagocytosis. IcsB has previously been demonstrated to be required for S. flexneri evasion of autophagy at late times during infection (4–6 hr) by inhibiting binding of the autophagy protein Atg5 to the Shigella surface protein IcsA (VirG). Our results suggest that IcsB and Toca-1 modulation of LC3 recruitment restricts LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants. Together with published results, our findings suggest that IcsB inhibits innate immune responses in two distinct ways, first, by inhibiting LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants early during infection, and second, by inhibiting autophagy late during infection. PMID:24722587

Human amyloidogenic light chain proteins result in cardiac dysfunction, cell death, and early mortality in zebrafish.

PubMed

Mishra, Shikha; Guan, Jian; Plovie, Eva; Seldin, David C; Connors, Lawreen H; Merlini, Giampaolo; Falk, Rodney H; MacRae, Calum A; Liao, Ronglih

2013-07-01

Systemic amyloid light-chain (AL) amyloidosis is associated with rapidly progressive and fatal cardiomyopathy resulting from the direct cardiotoxic effects of circulating AL light chain (AL-LC) proteins and the indirect effects of AL fibril tissue infiltration. Cardiac amyloidosis is resistant to standard heart failure therapies, and, to date, there are limited treatment options for these patients. The mechanisms underlying the development of cardiac amyloidosis and AL-LC cardiotoxicity are largely unknown, and their study has been limited by the lack of a suitable in vivo model system. Here, we establish an in vivo zebrafish model of human AL-LC-induced cardiotoxicity. AL-LC isolated from AL cardiomyopathy patients or control nonamyloidogenic LC protein isolated from multiple myeloma patients (Con-LC) was directly injected into the circulation of zebrafish at 48 h postfertilization. AL-LC injection resulted in impaired cardiac function, pericardial edema, and increased cell death relative to Con-LC, culminating in compromised survival with 100% mortality within 2 wk, independent of AL fibril deposition. Prior work has implicated noncanonical p38 MAPK activation in the pathogenesis of AL-LC-induced cardiotoxicity, and p38 MAPK inhibition via SB-203580 rescued AL-LC-induced cardiac dysfunction and cell death and attenuated mortality in zebrafish. This in vivo zebrafish model of AL-LC cardiotoxicity demonstrates that antagonism of p38 MAPK within the AL-LC cardiotoxic signaling response may serve to improve cardiac function and mortality in AL cardiomyopathy. Furthermore, this in vivo model system will allow for further study of the molecular underpinnings of AL cardiotoxicity and identification of novel therapeutic strategies.

Draft Genome Sequence of Lactobacillus crispatus EM-LC1, an Isolate with Antimicrobial Activity Cultured from an Elderly Subject

PubMed Central

Power, Susan E.; Harris, Hugh M. B.; Bottacini, Francesca; Ross, R. Paul; O’Toole, Paul W.

2013-01-01

Here we report the 1.86-Mb draft genome sequence of Lactobacillus crispatus EM-LC1, a fecal isolate with antimicrobial activity. This genome sequence is expected to provide insights into the antimicrobial activity of L. crispatus and improve our knowledge of its potential probiotic traits. PMID:24356836

Screening Antioxidants Using LC-MS: A Case Study with Cocoa

PubMed Central

Calderón, Angela I.; Wright, Brian J.; Hurst, W. Jeffrey; van Breemen, Richard B.

2009-01-01

Oxidative stress enhances pathological processes contributing to cancer, cardiovascular disease and neurodegenerative diseases, and dietary antioxidants may counteract these deleterious processes. Since rapid methods to evaluate and compare food products for antioxidant benefits are needed, a new assay based on liquid chromatography-mass spectrometry (LC-MS) was developed for the identification and quantitative analysis of antioxidants in complex natural product samples such as food extracts. This assay is based on the comparison of electrospray LC-MS profiles of sample extracts before and after treatment with reactive oxygen species such as hydrogen peroxide or DPPH (2,2-diphenyl-1-picrylhydrazyl radical). Using this assay, methanolic extracts of cocoa powder were analyzed, and procyanidins were found to be the most potent antioxidant species. These species were identified using LC-MS, LC-MS-MS, accurate mass measurement, and comparison with reference standards. Furthermore, LC-MS was used to determine the levels of these species in cocoa samples. Catechin and epicatechin were the most abundant antioxidants followed by their dimers and trimers. The most potent antioxidants in cocoa were trimers and dimers of catechin and epicatechin, such as procyanidin B2, followed by catechin and epicatechin. This new LC-MS assay facilitates the rapid identification and then the determination of the relative antioxidant activities of individual antioxidant species in complex natural product samples and food products such as cocoa. PMID:19489609

Photoresponsive smart surface of LC azo-dendrimer: photomanipulation of topological structures and real-time imaging at a nano-scale

NASA Astrophysics Data System (ADS)

Araoka, Fumito; Eremin, Alexey; Aya, Satoshi; Lee, Guksik; Ito, Atsuki; Nadasi, Hajnalka; Sebastian, Nerea; Ishikawa, Ken; Haba, Osamu; Stannarius, Ralf; Yonetake, Koichiro; Takezoe, Hideo

2017-02-01

In this paper, we review some results on our recent studies on photo-induced phenomena of liquid crystals (LCs) by means of interfaces decorated with a photo-responsive azobenzene dendrimer (azo-dendrimer). The azo-dendrimer molecules doped in a LC are spontaneously segregated from bulk and adsorbed onto substrate/LC or solvent/LC interfaces, and their photo-isomerization can bring about the so-called anchoring transition, i.e. reversible switching between homeotropic and planar alignment states of the bulk LC, when exposed to UV/VIS light. In addition to photoinduced anchoring transition in a LC cell, several interesting photo-induced phenomena through the azo-dendrimerdecorated interfaces have been reported, such as photo-induced transformation of the interior topological structures of nematic, cholesteric and smectic droplets, photo-mechanical motion of the micro particles dispersed in a nematic matrix, and optical assistance of the athermal anchoring transition with the aid of a perfluoropolymer surface. In addition to such phenomena, we also discuss the conditions of such photo-responsive interfaces in terms of the polar anchoring energy at the interface upon photo-isomerization under illumination of UV and/or VIS lights. The anisotropy of the polar anchoring energy was evaluated experimentally by means of Polarization Microscopy (POM), Dielectric Spectroscopy (DS), Second Harmonic Generation (SHG), and Attenuated Total Reflection Fourier Transform Infrared (ATR-IR) Spectroscopy, and theoretically based on the simple Rapini-Papoular model. We also demonstrate the continuous bulk orientation change by the photo-dynamic process through the fine control of the polar anchoring energy. Besides, the state-of-the-art video-rate atomic force microscopy (ν-AFM) was carried out to visualize the dynamics of such interfaces at a nano-meter scale.

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy.

PubMed

Sun, Aiqin; Wei, Jing; Childress, Chandra; Shaw, John H; Peng, Ke; Shao, Genbao; Yang, Wannian; Lin, Qiong

2017-03-04

The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.

IRE1α pathway of endoplasmic reticulum stress induces neuronal apoptosis in the locus coeruleus of rats under single prolonged stress.

PubMed

Zhao, Wei; Han, Fang; Shi, Yuxiu

2016-08-01

Our previous studies have shown evidence of endoplasmic reticulum (ER) stress-induced apoptosis in the hippocampus and mPFC in an animal model of post- traumatic stress disorder (PTSD). Inositol-requiring enzyme 1α (IRE1α) and its downstream molecule X-box binding protein 1 (XBP1) play key roles in the ER-related apoptosis pathway. Dysregulation of the locus coeruleus (LC) has been reported to contribute to cognitive and/or arousal impairments associated with PTSD. The aim of the present study was to explore the role of IRE1α pathway in neuronal apoptosis in the LC of rat models of PTSD. We used an acute exposure to prolonged stress (single prolonged stress, SPS) to model PTSD in rats and examined the effects related to the IRE1α pathway. Neuronal apoptosis in LC was detected by transmission electron microscopy and TUNEL staining. The results showed that the level of LC neuronal apoptosis was markedly increased after SPS. SPS exposure triggered IRE1α pathway, as evidenced by the increased activity of IRE1α, specific splicing of XBP1, and up-regulated expression of binding immunoglobulin protein/78kDa glucose-regulated protein (BiP/GRP78), and C/EBP-homologous protein (CHOP). Treatment with STF-083010, an IRE1α RNase-specific inhibitor, successfully attenuated the above changes. These results indicate that excessive activation of the ER stress-associated IRE1α pathway is involved in LC neuronal apoptosis induced by SPS exposure; this may be a crucial mechanism of the pathogenesis of PTSD. Copyright © 2016 Elsevier Inc. All rights reserved.

Species Identification of Bovine, Ovine and Porcine Type 1 Collagen; Comparing Peptide Mass Fingerprinting and LC-Based Proteomics Methods.

PubMed

Buckley, Mike

2016-03-24

Collagen is one of the most ubiquitous proteins in the animal kingdom and the dominant protein in extracellular tissues such as bone, skin and other connective tissues in which it acts primarily as a supporting scaffold. It has been widely investigated scientifically, not only as a biomedical material for regenerative medicine, but also for its role as a food source for both humans and livestock. Due to the long-term stability of collagen, as well as its abundance in bone, it has been proposed as a source of biomarkers for species identification not only for heat- and pressure-rendered animal feed but also in ancient archaeological and palaeontological specimens, typically carried out by peptide mass fingerprinting (PMF) as well as in-depth liquid chromatography (LC)-based tandem mass spectrometric methods. Through the analysis of the three most common domesticates species, cow, sheep, and pig, this research investigates the advantages of each approach over the other, investigating sites of sequence variation with known functional properties of the collagen molecule. Results indicate that the previously identified species biomarkers through PMF analysis are not among the most variable type 1 collagen peptides present in these tissues, the latter of which can be detected by LC-based methods. However, it is clear that the highly repetitive sequence motif of collagen throughout the molecule, combined with the variability of the sites and relative abundance levels of hydroxylation, can result in high scoring false positive peptide matches using these LC-based methods. Additionally, the greater alpha 2(I) chain sequence variation, in comparison to the alpha 1(I) chain, did not appear to be specific to any particular functional properties, implying that intra-chain functional constraints on sequence variation are not as great as inter-chain constraints. However, although some of the most variable peptides were only observed in LC-based methods, until the range of

Quantitation of melatonin and n-acetylserotonin in human plasma by nanoflow LC-MS/MS and electrospray LC-MS/MS.

PubMed

Carter, Melissa D; Calcutt, M Wade; Malow, Beth A; Rose, Kristie L; Hachey, David L

2012-03-01

Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100  mm, 3.5 µm) or on a polyimide-coated, fused-silica capillary self-packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7-1165  pg/mL, LC: 1165-116,500  pg/mL) and for NAS (nanoflow LC: 11.0-1095  pg/mL). Copyright © 2012 John Wiley & Sons, Ltd.

Quantitation of Melatonin and N-acetylserotonin in Human Plasma by Nanoflow LC-MS/MS and Electrospray LC-MS/MS

PubMed Central

Carter, Melissa D.; Calcutt, M. Wade; Malow, Beth A.; Rose, Kristie L.; Hachey, David L.

2012-01-01

Melatonin (MEL) and its chemical precursor N-acetylserotonin (NAS) are believed to be potential biomarkers for sleep-related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability, and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC-MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1×100 mm, 3.5 μm) or on a polyimide-coated, fused-silica capillary self-packed with 17 cm AquaC18 (3 μm, 125 Å). Quantitation was done using the SRM transitions m/z 233→174 and m/z 219→160 for MEL and NAS, respectively. The analytical response ratio vs. concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL). PMID:22431453

Dot-Blot Immunoassay of Fasciola gigantica Infection using 27 kDa and Adult Worm Regurge Antigens in Egyptian Patients

PubMed Central

Kamel, Hanan H.; Saad, Ghada A.

2013-01-01

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable. PMID:23710084

Dot-blot immunoassay of Fasciola gigantica infection using 27 kDa and adult worm regurge antigens in Egyptian patients.

PubMed

Kamel, Hanan H; Saad, Ghada A; Sarhan, Rania M

2013-04-01

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.

Heat shock 70-kDa protein 8 isoform 1 is expressed on the surface of human embryonic stem cells and downregulated upon differentiation.

PubMed

Son, Yeon Sung; Park, Jae Hyun; Kang, Young Kook; Park, Jin-Sung; Choi, Hong Seo; Lim, Ji Young; Lee, Jeoung Eun; Lee, Jung Bok; Ko, Myoung Seok; Kim, Yong-Sam; Ko, Jeong-Heon; Yoon, Hyun Soo; Lee, Kwang-Woong; Seong, Rho Hyun; Moon, Shin Yong; Ryu, Chun Jeih; Hong, Hyo Jeong

2005-01-01

The cell-surface markers used routinely to define the undifferentiated state and pluripotency of human embryonic stem cells (hESCs) are those used in mouse embryonic stem cells (mESCs) because of a lack of markers directly originated from hESC itself. To identify more hESC-specific cell-surface markers, we generated a panel of monoclonal antibodies (MAbs) by immunizing the irradiated cell clumps of hESC line Miz-hES1, and selected 26 MAbs that were able to bind to Miz-hES1 cells but not to mESCs, mouse embryonic fibroblast cells, and STO cells. Most antibodies did not bind to human neural progenitor cells derived from the Miz-hES1 cells, either. Of these, MAb 20-202S (IgG1, kappa) immunoprecipitated a cell-surface protein of 72-kDa from the lysate of biotin-labeled Miz-hES1 cells, which was identified to be heat shock 70-kDa protein 8 isoform 1 (HSPA8) by quadrupole time-of-flight tandem mass spectrometry. Immunocytochemical analyses proved that the HSPA8 protein was also present on the surface of hESC lines Miz-hES4, Miz-hES6, and HSF6. Two-color flow cytometric analysis of Miz-hES1 and HSF6 showed the coexpression of the HSPA8 protein with other hESC markers such as stage-specific embryonic antigen 3 (SSEA3), SSEA4, TRA-1-60, and TRA-1-81. Flow cytometric and Western blot analyses using various cells showed that MAb 20-202S specifically bound to the HSPA8 protein on the surface of Miz-hES1, contrary to other anti-HSP70 antibodies examined. Furthermore, the surface expression of the HSPA8 protein on Miz-hES1 was markedly downregulated upon differentiation. These data indicate that a novel MAb 20-202S recognizes the HSPA8 protein on the surface of hESCs and suggest that the HSPA8 protein is a putative cell-surface marker for undifferentiated hESCs.

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy

PubMed Central

Sun, Aiqin; Wei, Jing; Childress, Chandra; Shaw, John H.; Peng, Ke; Shao, Genbao; Yang, Wannian; Lin, Qiong

2017-01-01

ABSTRACT The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy. PMID:28085563

Qualitative and Quantitative Analysis of Proteome and Peptidome of Human Follicular Fluid Using Multiple Samples from Single Donor with LC-MS and SWATH Methodology.

PubMed

Lewandowska, Aleksandra E; Macur, Katarzyna; Czaplewska, Paulina; Liss, Joanna; Łukaszuk, Krzysztof; Ołdziej, Stanisław

2017-08-04

Human follicular fluid (hFF) is a natural environment of oocyte maturation, and some components of hFF could be used to judge oocyte capability for fertilization and further development. In our pilot small-scale study three samples from four donors (12 samples in total) were analyzed to determine which hFF proteins/peptides could be used to differentiate individual oocytes and which are patient-specific. Ultrafiltration was used to fractionate hFF to high-molecular-weight (HMW) proteome (>10 kDa) and low-molecular-weight (LMW) peptidome (<10 kDa) fractions. HMW and LMW compositions were analyzed using LC-MS in SWATH data acquisition and processing methodology. In total we were able to identify 158 proteins, from which 59 were never reported before as hFF components. 55 (45 not reported before) proteins were found by analyzing LMW fraction, 67 (14 not reported before) were found by analyzing HMW fraction, and 36 were identified in both fractions of hFF. We were able to perform quantitative analysis for 72 proteins from HMW fraction of hFF. We found that concentrations of 11 proteins varied substantially among hFF samples from single donors, and those proteins are promising targets to identify biomarkers useful in oocyte quality assessment.

Evaluation of the Light-Cycler® SeptiFast Test in Newborns With Suspicion of Nosocomial Sepsis

PubMed Central

Ortiz Ibarra, Javier; Trevino Valdez, Pablo; Valenzuela Mendez, Ema; Limon Rojas, Ana; Lara Flores, Gabriel; Ceballos Bocanegra, Adrian; Morales Mendez, Iyari; Fernandez Carrocera, Luis; Covian Molina, Emilia; Reyna Figueroa, Jesus

2015-01-01

Background: Nosocomial sepsis (NS) in newborns (NBs) is associated with high mortality rates and low microbial recovery rates. To overcome the latter problem, new techniques in molecular biology are being used. Objectives: To evaluate the diagnostic efficacy of SeptiFast test for the diagnosis of nosocomial sepsis in the newborn. Materials and Methods: 86 blood specimens of NBs with suspected NS (NOSEP-1 Test > 8 points) were analyzed using Light Cycler SeptiFast (LC-SF) a real-time multiplex PCR instrument. The results were analyzed with the Roche SeptiFast Identification Software. Another blood sample was collected to carry out a blood culture (BC). Results: Sensitivity (Sn) and specificity (Sp) of 0.69 and 0.65 respectively, compared with blood culture (BC) were obtained for LC-SF. Kappa index concordance between LC-SF and BC was 0.21. Thirteen (15.11%) samples were BC positive and 34 (31.39%) were positive with LC-SF tests. Conclusions: Compared with BC, LC-SF allows the detection of a greater number of pathogenic species in a small blood sample (1 mL) with a shorter response time. PMID:26199693

Characterization of a mixture of lobster digestive cysteine proteinases by ionspray mass spectrometry and tryptic mapping with LC--MS and LC--MS--MS

NASA Astrophysics Data System (ADS)

Thibault, P.; Pleasance, S.; Laycock, M. V.; Mackay, R. M.; Boyd, R. K.

1991-12-01

An inseparable mixture of two cysteine proteinases, isolated from the digestive tract of the American lobster, was investigated by ionspray mass spectrometry (ISP-MS), using a combination of infusion of intact proteins with on-line liquid chromatography--mass spectrometry (LC--MS) and LC--MS--MS analyses of tryptic digests. These data were interpreted by comparisons with predictions from results of molecular cloning of cysteine-proteinase-encoding messenger RNA sequences previously isolated from the lobster hepatopancreas. Investigations of the numbers of free thiol groups and of disulfide bonds were made by measuring the molecular weights of the alkylated proteins with and without prior reduction of disulfide bonds, and comparison with the corresponding data for the native proteins. Identification of tyrptic fragment peptides containing cysteine residues was facilitated by comparing LC--MS analyses of tryptic digests of denatured and of denatured and alkylated proteins, since such tryptic peptides are subject to shifts in both mass and retention time upon reduction and alkylation. Confirmation of amino acid sequences was obtained from fragment ion spectra of each tryptic peptide (alkylated or not) as it eluted from the column. Acquisition of such on-line LC--MS data was possible through use of the entire effluent from a standard 1 mm high performance liquid chromatography (HPLC) column by an IonsSpray® LC--MS interface (pneumatically assisted electrospray).

Phototropin 1 and dim-blue light modulate the red light de-etiolation response.

PubMed

Wang, Yihai; M Folta, Kevin

2014-01-01

Light signals regulate seedling morphological changes during de-etiolation through the coordinated actions of multiple light-sensing pathways. Previously we have shown that red-light-induced hypocotyl growth inhibition can be reversed by addition of dim blue light through the action of phototropin 1 (phot1). Here we further examine the fluence-rate relationships of this blue light effect in short-term (hours) and long-term (days) hypocotyl growth assays. The red stem-growth inhibition and blue promotion is a low-fluence rate response, and blue light delays or attenuates both the red light and far-red light responses. These de-etiolation responses include blue light reversal of red or far-red induced apical hook opening. This response also requires phot1. Cryptochromes (cry1 and cry2) are activated by higher blue light fluence-rates and override phot1's influence on hypocotyl growth promotion. Exogenous application of auxin transport inhibitor naphthylphthalamic acid abolished the blue light stem growth promotion in both hypocotyl growth and hook opening. Results from the genetic tests of this blue light effect in auxin transporter mutants, as well as phytochrome kinase substrate mutants indicated that aux1 may play a role in blue light reversal of red light response. Together, the phot1-mediated adjustment of phytochrome-regulated photomorphogenic events is most robust in dim blue light conditions and is likely modulated by auxin transport through its transporters.

Phototropin 1 and dim-blue light modulate the red light de-etiolation response

PubMed Central

Wang, Yihai; M Folta, Kevin

2014-01-01

Light signals regulate seedling morphological changes during de-etiolation through the coordinated actions of multiple light-sensing pathways. Previously we have shown that red-light-induced hypocotyl growth inhibition can be reversed by addition of dim blue light through the action of phototropin 1 (phot1). Here we further examine the fluence-rate relationships of this blue light effect in short-term (hours) and long-term (days) hypocotyl growth assays. The red stem-growth inhibition and blue promotion is a low-fluence rate response, and blue light delays or attenuates both the red light and far-red light responses. These de-etiolation responses include blue light reversal of red or far-red induced apical hook opening. This response also requires phot1. Cryptochromes (cry1 and cry2) are activated by higher blue light fluence-rates and override phot1's influence on hypocotyl growth promotion. Exogenous application of auxin transport inhibitor naphthylphthalamic acid abolished the blue light stem growth promotion in both hypocotyl growth and hook opening. Results from the genetic tests of this blue light effect in auxin transporter mutants, as well as phytochrome kinase substrate mutants indicated that aux1 may play a role in blue light reversal of red light response. Together, the phot1-mediated adjustment of phytochrome-regulated photomorphogenic events is most robust in dim blue light conditions and is likely modulated by auxin transport through its transporters. PMID:25482790

Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples

USDA-ARS?s Scientific Manuscript database

High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-...

Interaction of human platelets with laminin and identification of the 67 kDa laminin receptor on platelets.

PubMed Central

Tandon, N N; Holland, E A; Kralisz, U; Kleinman, H K; Robey, F A; Jamieson, G A

1991-01-01

A microtitre adhesion assay has been developed to define parameters affecting the adherence of washed platelets to laminin. Adherence was optimally supported by Mg2+ and was inhibited by Ca2+ and by anti-laminin Fab fragments, but significant adhesion (75-90% of control) was found both in heparinized plasma containing physiological levels of bivalent cations and in plasma anti-coagulated with EGTA. Adherence was unaffected by platelet activation with ADP but was decreased by 50% by treatment with alpha-thrombin (1 unit/ml, 5 min). Adherence was unaffected by monospecific polyclonal antibodies to glycoprotein (GP) Ib and GPIV, and was normal with platelets from two patients with Glanzmann's thrombasthaenia, indicating that GPIb, the GPIIb/IIIa complex and GPIV are not involved in platelet-laminin interaction. Affinity chromatography of Triton-solubilized membranes on laminin-Sepharose followed by elution with 0.2 M-glycine/HCl (pH 2.85) identified a major band with a molecular mass of 67 kDa in the reduced and of 53 kDa in the unreduced form. This protein gave a positive reaction on Western blotting with a monospecific polyclonal antibody raised against the high-affinity laminin receptor isolated from human breast carcinoma tissue. The adhesion of platelets to laminin was inhibited by two monoclonal IgM antibodies specific to the LR-1 domain of the 67 kDa receptor. The binding protein was surface-oriented, as shown by flow cytofluorimetry and by the fact that it could be iodinated in intact platelets, but it was not labelled by the periodate-borotritide procedure, suggesting that it did not contain terminal sialic acid. The laminin-derived peptides Tyr-Ile-Gly-Ser-Arg and Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg-NH2, which constitute a complementary binding domain in laminin for the 67 kDa receptor, themselves supported platelet adhesion, bound to the receptor and inhibited the adhesion of platelets to laminin. In addition, Fab fragments of anti

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

PubMed

Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

The blue light-induced interaction of cryptochrome 1 with COP1 requires SPA proteins during Arabidopsis light signaling.

PubMed

Holtkotte, Xu; Ponnu, Jathish; Ahmad, Margaret; Hoecker, Ute

2017-10-01

Plants constantly adjust their growth, development and metabolism to the ambient light environment. Blue light is sensed by the Arabidopsis photoreceptors CRY1 and CRY2 which subsequently initiate light signal transduction by repressing the COP1/SPA E3 ubiquitin ligase. While the interaction between cryptochromes and SPA is blue light-dependent, it was proposed that CRY1 interacts with COP1 constitutively, i.e. also in darkness. Here, our in vivo co-immunoprecipitation experiments suggest that CRY1 and CRY2 form a complex with COP1 only after seedlings were exposed to blue light. No association between COP1 and CRY1 or CRY2 was observed in dark-grown seedlings. Thus, our results suggest that cryptochromes bind the COP1/SPA complex after photoactivation by blue light. In a spa quadruple mutant that is devoid of all four SPA proteins, CRY1 and COP1 did not interact in vivo, neither in dark-grown nor in blue light-grown seedlings. Hence, SPA proteins are required for the high-affinity interaction between CRY1 and COP1 in blue light. Yeast three-hybrid experiments also show that SPA1 enhances the CRY1-COP1 interaction. The coiled-coil domain of SPA1 which is responsible for COP1-binding was necessary to mediate a CRY1-SPA1 interaction in vivo, implying that-in turn-COP1 may be necessary for a CRY1-SPA1 complex formation. Hence, SPA1 and COP1 may act cooperatively in recognizing and binding photoactivated CRY1. In contrast, the blue light-induced association between CRY2 and COP1 was not dependent on SPA proteins in vivo. Similarly, ΔCC-SPA1 interacted with CRY2, though with a much lower affinity than wild-type SPA1. In total, our results demonstrate that CRY1 and CRY2 strongly differ in their blue light-induced interaction with the COP1/SPA complex.

Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

PubMed

Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

2016-10-01

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

Occulting Light Concentrators in Liquid Scintillator Neutrino Detectors

NASA Astrophysics Data System (ADS)

Buizza Avanzini, Margherita; Cabrera, Anatael; Dusini, Stefano; Grassi, Marco; He, Miao; Wu, Wenjie

2017-09-01

The experimental efforts characterizing the era of precision neutrino physics revolve around collecting high-statistics neutrino samples and attaining an excellent energy and position resolution. Next generation liquid-based neutrino detectors, such as JUNO, HyperKamiokande, etc, share the use of a large target mass, and the need of pushing light collection to the edge for maximal calorimetric information. Achieving high light collection implies considerable costs, especially when considering detector masses of several kt. A traditional strategy to maximize the effective photo-coverage with the minimum number of PMTs relies on Light Concentrators (LC), such as Winston Cones. In this paper, the authors introduce a novel concept called Occulting Light Concentrators (OLC), whereby a traditional LC gets tailored to a conventional PMT, by taking into account its single-photoelectron collection efficiency profile and thus occulting the worst performing portion of the photocathode. Thus, the OLC shape optimization takes into account not only the optical interface of the PMT, but also the maximization of the PMT detection performances. The light collection uniformity across the detector is another advantage of the OLC system. By considering the case of JUNO, we will show OLC capabilities in terms of light collection and energy resolution.

Melanoma cultures show different susceptibility towards E1A-, E1B-19 kDa- and fiber-modified replication-competent adenoviruses.

PubMed

Schmitz, M; Graf, C; Gut, T; Sirena, D; Peter, I; Dummer, R; Greber, U F; Hemmi, S

2006-06-01

Replicating adenovirus (Ad) vectors with tumour tissue specificity hold great promise for treatment of cancer. We have recently constructed a conditionally replicating Ad5 AdDeltaEP-TETP inducing tumour regression in a xenograft mouse model. For further improvement of this vector, we introduced four genetic modifications and analysed the viral cytotoxicity in a large panel of melanoma cell lines and patient-derived melanoma cells. (1) The antiapoptotic gene E1B-19 kDa (Delta19 mutant) was deleted increasing the cytolytic activity in 18 of 21 melanoma cells. (2) Introduction of the E1A 122-129 deletion (Delta24 mutant), suggested to attenuate viral replication in cell cycle-arrested cells, did not abrogate this activity and increased the cytolytic activity in two of 21 melanoma cells. (3) We inserted an RGD sequence into the fiber to extend viral tropism to alphav integrin-expressing cells, and (4) swapped the fiber with the Ad35 fiber (F35) enhancing the tropism to malignant melanoma cells expressing CD46. The RGD-fiber modification strongly increased cytolysis in all of the 11 CAR-low melanoma cells. The F35 fiber-chimeric vector boosted the cytotoxicity in nine of 11 cells. Our results show that rational engineering additively enhances the cytolytic potential of Ad vectors, a prerequisite for the development of patient-customized viral therapies.

Molecular and Structural Traits of Insulin Receptor Substrate 1/LC3 Nuclear Structures and Their Role in Autophagy Control and Tumor Cell Survival.

PubMed

Lassak, Adam; Dean, Mathew; Wyczechowska, Dorota; Wilk, Anna; Marrero, Luis; Trillo-Tinoco, Jimena; Boulares, A Hamid; Sarkaria, Jann N; Del Valle, Luis; Peruzzi, Francesca; Ochoa, Augusto; Reiss, Krzysztof

2018-05-15

Insulin receptor substrate 1 (IRS-1) is a common cytosolic adaptor molecule involved in signal transduction from insulin and insulin-like growth factor I (IGF-I) receptors. IRS-1 can also be found in the nucleus. We report here a new finding of unique IRS-1 nuclear structures, which we observed initially in glioblastoma biopsy specimens and glioblastoma xenografts. These nuclear structures can be reproduced in vitro by the ectopic expression of IRS-1 cDNA cloned in frame with the nuclear localization signal (NLS-IRS-1). In these structures, IRS-1 localizes at the periphery, while the center harbors a key autophagy protein, LC3. These new nuclear structures are highly dynamic, rapidly exchange IRS-1 molecules with the surrounding nucleoplasm, disassemble during mitosis, and require a growth stimulus for their reassembly and maintenance. In tumor cells engineered to express NLS-IRS-1, the IRS-1/LC3 nuclear structures repress autophagy induced by either amino acid starvation or rapamycin treatment. In this process, IRS-1 nuclear structures sequester LC3 inside the nucleus, possibly preventing its cytosolic translocation and the formation of new autophagosomes. This novel mechanism provides a quick and reversible way of inhibiting autophagy, which could counteract autophagy-induced cancer cell death under severe stress, including anticancer therapies. Copyright © 2018 American Society for Microbiology.

Submillisecond-response IR spatial light modulators with polymer network liquid crystal

NASA Astrophysics Data System (ADS)

Sun, Jie; Chen, Yuan; Wu, Shin-Tson

2013-03-01

Polymer network liquid crystal (PNLC) is attractive for many photonic applications because of its fast response time and large phase modulation. However, the voltage-on state light scattering caused by multi-domains of LC molecules hinders its applications in the visible and near infrared regions. To reduce domain sizes and eliminate scattering for λ=1.06 μm and 1.55 μm, we studied the effect of LC viscosity on domain sizes. PNLCs based on five different LC hosts were prepared. The LC host was first mixed with 6% reactive mesogen and then filled into a 12-μm cell with homogeneous alignment. After UV curing, we measured the on-state transmission spectra of these five PNLCs. By fitting the transmission spectra with Rayleigh-Gans-Debye model, we can estimate the average domain sizes. We found that the domain sizes of PNLC are inversely proportional to the rotational viscosity of the LC host. This finding can be explained by the Stokes-Einstein equation. As a result, PNLC with a slower diffusion rate would cause smaller domain sizes, which in turn lead to faster response time. To achieve a slower diffusion rate, we cured the PNLC samples at a lower temperature. By selecting a high viscosity and high Δɛ LC host, we demonstrate a scattering-free (<3%) 2π phase modulator at λ=1.06 μm and λ=1.55 μm. Temperature affects the PNLC performance significantly. As the operation temperature increases from 25oC to 70oC, the response time drops from 220 μs to 30 μs. 2π operating voltage for λ=1.06 μm slightly increases from 65V to 85V. Meanwhile, hysteresis decreases from 7.7% to 2%. For λ=1.55μm, operating voltage is 100V. If reflective mode is employed, operating voltage can be reduced to 55V.

Microparticles prepared with 50-190kDa chitosan as promising non-toxic carriers for pulmonary delivery of isoniazid.

PubMed

Oliveira, Paula M; Matos, Breno N; Pereira, Priscilla A T; Gratieri, Taís; Faccioli, Lucia H; Cunha-Filho, Marcílio S S; Gelfuso, Guilherme M

2017-10-15

Chitosan biocompatibility and mucoadhesiveness make it an ideal polymer for antituberculotic drugs microcapsulation for pulmonary delivery. Yet, previous study indicated toxicity problems to J-774.1-cells treated with some medium molecular weight (190-310kDa) chitosan microparticles. As polymer molecular weight is a crucial factor to be considered, this paper describes the preparation and characterization of chitosan (50-190kDa) microparticles containing isoniazid (INH). Cytotoxicity assays were also performed on murine peritoneal (J-774.1) and alveolar (AMJ2-C11) macrophages cell lines, followed by cytokines detection from AMJ2-C11 cells. Spray-drying process produced mucoadhesive microparticles from 3.2μm to 3.9μm, entrapping more than 89% of the drug and preserving their chemical stability. Drug release behavior could be controlled by the use of cross-linked or uncross-linked chitosan, the latter leading to a rapid drug release. Mucoadhesive potential of the microparticles was characterized following in vitro and ex vivo assays. Finally, a significant reduction on toxicity against peritoneal macrophages and no toxic effect on alveolar macrophages with use of such microparticles were observed. In conclusion, 50-190kDa chitosan microparticles may act as promising non-cytotoxic carriers for pulmonary delivery of INH showing marked alveoli macrophage activation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tryptic digestion of human GPIIIa. Isolation and biochemical characterization of the 23 kDa N-terminal glycopeptide carrying the antigenic determinant for a monoclonal antibody (P37) which inhibits platelet aggregation.

PubMed Central

Calvete, J J; Rivas, G; Maruri, M; Alvarez, M V; McGregor, J L; Hew, C L; Gonzalez-Rodriguez, J

1988-01-01

Early digestion of pure human platelet glycoprotein IIIa (GPIIIa) leads to a single cleavage of the molecule at 23 kDa far from one of the terminal amino acids. Automated Edman degradation demonstrates that GPIIIa and the smaller (23 kDa) tryptic fragment share the same N-terminal amino acid sequence. A further cleavage occurs in the larger fragment (80 kDa), reducing its apparent molecular mass by 10 kDa. The 23 kDa fragment remains attached to the larger ones in unreduced samples. Stepwise reduction of early digested GPIIIa with dithioerythritol selectively reduces the single disulphide bond joining the smaller (23 kDa) to the larger (80/70 kDa) fragments. Two fractions were obtained by size-exclusion chromatography of early digested GPIIIa after partial or full reduction and alkylation. The larger-size fraction contains the 80/70 kDa fragments, while the 23 kDa fragment is isolated in the smaller. The amino acid compositions of these fractions do not differ very significantly from the composition of GPIIIa; however the 23 kDa fragment contains only 10.2% by weight of sugars and is richer in neuraminic acid. Disulphide bonds are distributed four in the 23 kDa glycopeptide and 20-21 in the 80/70 kDa glycopeptide. The epitope for P37, a monoclonal antibody which inhibits platelet aggregation [Melero & González-Rodríguez (1984) Eur. J. Biochem. 141, 421-427] is situated within the first 17 kDa of the N-terminal region of GPIIIa, which gives a special functional interest to this extracellular region of GPIIIa. On the other hand, the epitopes for GPIIIa-specific monoclonal antibodies, P6, P35, P40 and P97, which do not interfere with platelet aggregation, are located within the larger tryptic fragment (80/70 kDa). Thus, the antigenic areas available in the extracellular surface of GPIIIa for these five monoclonal antibodies are now more precisely delineated. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2455507

In vivo exposure to ozone produces an increase in a 72-kDa heat shock protein in guinea pigs.

PubMed

Su, W Y; Gordon, T

1997-09-01

Although several lines of evidence have suggested that oxidizing agents can induce heat shock proteins (HSPs) in vitro, little is known about the induction of HSPs during in vivo exposure to oxidants. Guinea pigs were exposed to ozone for 6 h and euthanized up to 72 h later. Proteins from lavage cells and lung tissue were characterized by immunoblotting with 72- and 73/72-kDa HSP monoclonal antibodies. Although 73-kDa HSP was expressed constituitively in lung tissue, it was not affected by ozone. In contrast, 72-kDa HSP was significantly increased in lavage cells and lung tissue of animals exposed to 0.4 and 0.66 parts/million of ozone. Both heat treatment and arsenite induced 72-kDa HSP in cultured alveolar macrophages. The increase in 72-kDa HSP in the lavage cell pellet peaked at 24 h after ozone, whereas the influx of polymorphonuclear leukocytes peaked at 4 h. Examination of the induction of HSPs by ozone may provide clues to the development of ozone tolerance in humans and animals.

Cloning and sequencing of a gene encoding the 69-kDa extracellular chitinase of Janthinobacterium lividum.

PubMed

Gleave, A P; Taylor, R K; Morris, B A; Greenwood, D R

1995-09-15

Janthinobacterium lividum secretes a major 56-kDa chitinase and a minor 69-kDa chitinase. A chitinase gene was defined on a 3-kb fragment of clone pRKT10, by virtue of fluorescent colonies in the presence of 4-methylumbelliferyl-beta-D-N,N',N"-chitotrioside. Nucleotide sequencing revealed an 1998-bp open reading frame with the potential to encode a 69,716-Da protein with amino acid sequences similar to those in other chitinases, suggesting it encodes the minor chitinase (Chi69). Chitinase activity of Escherichia coli (pRKT10) lysates was detected mainly in the periplasmic fraction and immunoblotting detected a 70-kDa protein in this fraction. Chi69 has an N-terminal secretory leader peptide preceding two probable chitin-binding domains and a catalytic domain. These functional domains are separated by linker regions of proline-threonine repeats. Amino acid sequencing of cyanogen bromide cleavage-derived peptides from the major 56-kDa chitinase suggested that Chi69 may be a precursor of Chi56. In addition, an N-terminally truncated version of Chi69 retained chitinase activity as expected if in vivo processing of Chi69 generates Chi56.

Clinical Utility of LC3 and p62 Immunohistochemistry in Diagnosis of Drug-Induced Autophagic Vacuolar Myopathies: A Case-Control Study

PubMed Central

Lee, Han S.; Daniels, Brianne H.; Salas, Eduardo; Bollen, Andrew W.; Debnath, Jayanta; Margeta, Marta

2012-01-01

Background Some patients treated with chloroquine, hydroxychloroquine, or colchicine develop autophagic vacuolar myopathy, the diagnosis of which currently requires electron microscopy. The goal of the current study was to develop an immunohistochemical diagnostic marker for this pathologic entity. Methodology Microtubule-associated protein light chain 3 (LC3) has emerged as a robust marker of autophagosomes. LC3 binds p62/SQSTM1, an adapter protein that is selectively degraded via autophagy. In this study, we evaluated the utility of immunohistochemical stains for LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar myopathy. The staining was performed on archival muscle biopsy material, with subject assignment to normal control, drug-treated control, and autophagic myopathy groups based on history of drug use and morphologic criteria. Principal Findings In all drug-treated subjects, but not in normal controls, LC3 and p62 showed punctate staining characteristic of autophagosome buildup. In the autophagic myopathy subjects, puncta were coarser and tended to coalesce into linear structures aligned with the longitudinal axis of the fiber, often in the vicinity of vacuoles. The percentage of LC3- and p62-positive fibers was significantly higher in the autophagic myopathy group compared to either the normal control (p<0.001) or the drug-treated control group (p<0.05). With the diagnostic threshold set between 8% and 15% positive fibers (depending on the desired level of sensitivity and specificity), immunohistochemical staining for either LC3 or p62 could be used to identify subjects with autophagic vacuolar myopathy within the drug-treated subject group (p≤0.001). Significance Immunohistochemistry for LC3 and p62 can facilitate tissue-based diagnosis of drug-induced autophagic vacuolar myopathies. By limiting the need for electron microscopy (a time consuming and costly technique with high specificity, but low sensitivity), clinical use of these

Clinical utility of LC3 and p62 immunohistochemistry in diagnosis of drug-induced autophagic vacuolar myopathies: a case-control study.

PubMed

Lee, Han S; Daniels, Brianne H; Salas, Eduardo; Bollen, Andrew W; Debnath, Jayanta; Margeta, Marta

2012-01-01

Some patients treated with chloroquine, hydroxychloroquine, or colchicine develop autophagic vacuolar myopathy, the diagnosis of which currently requires electron microscopy. The goal of the current study was to develop an immunohistochemical diagnostic marker for this pathologic entity. Microtubule-associated protein light chain 3 (LC3) has emerged as a robust marker of autophagosomes. LC3 binds p62/SQSTM1, an adapter protein that is selectively degraded via autophagy. In this study, we evaluated the utility of immunohistochemical stains for LC3 and p62 as diagnostic markers of drug-induced autophagic vacuolar myopathy. The staining was performed on archival muscle biopsy material, with subject assignment to normal control, drug-treated control, and autophagic myopathy groups based on history of drug use and morphologic criteria. In all drug-treated subjects, but not in normal controls, LC3 and p62 showed punctate staining characteristic of autophagosome buildup. In the autophagic myopathy subjects, puncta were coarser and tended to coalesce into linear structures aligned with the longitudinal axis of the fiber, often in the vicinity of vacuoles. The percentage of LC3- and p62-positive fibers was significantly higher in the autophagic myopathy group compared to either the normal control (p<0.001) or the drug-treated control group (p<0.05). With the diagnostic threshold set between 8% and 15% positive fibers (depending on the desired level of sensitivity and specificity), immunohistochemical staining for either LC3 or p62 could be used to identify subjects with autophagic vacuolar myopathy within the drug-treated subject group (p ≤ 0.001). Immunohistochemistry for LC3 and p62 can facilitate tissue-based diagnosis of drug-induced autophagic vacuolar myopathies. By limiting the need for electron microscopy (a time consuming and costly technique with high specificity, but low sensitivity), clinical use of these markers will improve the speed and accuracy of

Crystal structure of the 25 kDa subunit of human cleavage factor Im

PubMed Central

Coseno, Molly; Martin, Georges; Berger, Christopher; Gilmartin, Gregory; Keller, Walter; Doublié, Sylvie

2008-01-01

Cleavage factor Im is an essential component of the pre-messenger RNA 3′-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein–protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic α/β/α fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Å, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3′-end processing. PMID:18445629

NIR-Vis-UV Light-Responsive Actuator Films of Polymer-Dispersed Liquid Crystal/Graphene Oxide Nanocomposites.

PubMed

Cheng, Zhangxiang; Wang, Tianjie; Li, Xiao; Zhang, Yihe; Yu, Haifeng

2015-12-16

To take full advantage of sunlight for photomechanical materials, NIR-vis-UV light-responsive actuator films of polymer-dispersed liquid crystal (PDLC)/graphene oxide (GO) nanocomposites were fabricated. The strategy is based on phase transition of LCs from nematic to isotropic phase induced by combination of photochemical and photothermal processes in the PDLC/GO nanocomposites. Upon mechanical stretching of the film, both topological shape change and mesogenic alignment occurred in the separated LC domains, enabling the film to respond to NIR-vis-UV light. The homodispersed GO flakes act as photoabsorbent and nanoscale heat source to transfer NIR or VIS light into thermal energy, heating the film and photothermally inducing phase transition of LC microdomains. By utilizing photochemical phase transition of LCs upon UV-light irradiation, one azobenzene dye was incorporated into the LC domains, endowing the nanocomposite films with UV-responsive property. Moreover, the light-responsive behaviors can be well-controlled by adjusting the elongation ratio upon mechanical treatment. The NIR-vis-UV light-responsive PDLC/GO nanocomposite films exhibit excellent properties of easy fabrication, low-cost, and good film-forming and mechanical features, promising their numerous applications in the field of soft actuators and optomechanical systems driven directly by sunlight.

Chip-LC-MS for label-free profiling of human serum.

PubMed

Horvatovich, Peter; Govorukhina, Natalia I; Reijmers, Theo H; van der Zee, Ate G J; Suits, Frank; Bischoff, Rainer

2007-12-01

The discovery of biomarkers in easily accessible body fluids such as serum is one of the most challenging topics in proteomics requiring highly efficient separation and detection methodologies. Here, we present the application of a microfluidics-based LC-MS system (chip-LC-MS) to the label-free profiling of immunodepleted, trypsin-digested serum in comparison to conventional capillary LC-MS (cap-LC-MS). Both systems proved to have a repeatability of approximately 20% RSD for peak area, all sample preparation steps included, while repeatability of the LC-MS part by itself was less than 10% RSD for the chip-LC-MS system. Importantly, the chip-LC-MS system had a two times higher resolution in the LC dimension and resulted in a lower average charge state of the tryptic peptide ions generated in the ESI interface when compared to cap-LC-MS while requiring approximately 30 times less (~5 pmol) sample. In order to characterize both systems for their capability to find discriminating peptides in trypsin-digested serum samples, five out of ten individually prepared, identical sera were spiked with horse heart cytochrome c. A comprehensive data processing methodology was applied including 2-D smoothing, resolution reduction, peak picking, time alignment, and matching of the individual peak lists to create an aligned peak matrix amenable for statistical analysis. Statistical analysis by supervised classification and variable selection showed that both LC-MS systems could discriminate the two sample groups. However, the chip-LC-MS system allowed to assign 55% of the overall signal to selected peaks against 32% for the cap-LC-MS system.

Bim Inhibits Autophagy by Recruiting Beclin 1 to Microtubules

PubMed Central

Luo, Shouqing; Garcia-Arencibia, Moises; Zhao, Rui; Puri, Claudia; Toh, Pearl P.C.; Sadiq, Oana; Rubinsztein, David C.

2012-01-01

Summary Bim is a proapoptotic BH3-only Bcl-2 family member. In response to death stimuli, Bim dissociates from the dynein light chain 1 (DYNLL1/LC8), where it is inactive, and can then initiate Bax/Bak-mediated mitochondria-dependent apoptosis. We found that Bim depletion increases autophagosome synthesis in cells and in vivo, and this effect is inhibited by overexpression of cell death-deficient Bim. Bim inhibits autophagy by interacting with Beclin 1, an autophagy regulator, and this interaction is facilitated by LC8. Bim bridges the Beclin 1-LC8 interaction and thereby inhibits autophagy by mislocalizing Beclin 1 to the dynein motor complex. Starvation, an autophagic stimulus, induces Bim phosphorylation, which abrogates LC8 binding to Bim, leading to dissociation of Bim and Beclin 1. Our data suggest that Bim switches locations between apoptosis-inactive/autophagy-inhibitory and apoptosis-active/autophagy-permissive sites. PMID:22742832

The WD40 domain of ATG16L1 is required for its non-canonical role in lipidation of LC3 at single membranes.

PubMed

Fletcher, Katherine; Ulferts, Rachel; Jacquin, Elise; Veith, Talitha; Gammoh, Noor; Arasteh, Julia M; Mayer, Ulrike; Carding, Simon R; Wileman, Thomas; Beale, Rupert; Florey, Oliver

2018-02-15

A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double-membrane phagophore, which is driven by the ATG16L1/ATG5-ATG12 complex. In contrast, non-canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3-associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non-canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat-containing C-terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non-canonical autophagy specifically, we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD Further, we demonstrate activation of non-canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non-canonical use of autophagy machinery. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Identification of a 27.8 kDa protein from flounder gill cells involved in lymphocystis disease virus binding and infection.

PubMed

Wang, Mu; Sheng, Xiu-Zhen; Xing, Jing; Tang, Xiao-Qian; Zhan, Wen-Bin

2011-03-16

In vitro, lymphocystis disease virus (LCDV) infection of flounder gill (FG) cell cultures causes obvious cytopathic effect (CPE). We describe attempts to isolate and characterize the LCDV-binding molecule(s) on the plasma membrane of FG cells that were responsible for virus entry. The results showed that the co-immunoprecipitation assay detected a 27.8 kDa molecule from FG cells that bound to LCDV. In a blocking ELISA, pre-incubation of FG cell membrane proteins with the specific antiserum developed against the 27.8 kDa protein could block LCDV binding. Similarly, antiserum against 27.8 kDa protein could also inhibit LCDV infection of FG cells in vitro. Mass spectrometric analysis established that the 27.8 kDa protein and beta-actin had a strong association. These results strongly supported the possibility that the 27.8 kDa protein was the putative receptor specific for LCDV infection of FG cells.

1. Light tower/keeper's house and abandoned light tower, view northwest, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

1. Light tower/keeper's house and abandoned light tower, view northwest, south southeast and east northeast sides - Matinicus Rock Light Station, Matinicus Island, on Matinicus Rock, Matinicus, Knox County, ME

Effect of first dimension phase selectivity in online comprehensive two dimensional liquid chromatography (LC × LC)

PubMed Central

Gu, Haiwei; Huang, Yuan; Filgueira, Marcelo; Carr, Peter W.

2012-01-01

In this study, we examined the effect of first dimension column selectivity in reversed phase (RP) online comprehensive two dimensional liquid chromatography (LC × LC). The second dimension was always a carbon clad metal oxide reversed phase material. The hydrophobic subtraction model (HSM) and the related phase selective triangles were used to guide the selection of six different RP first dimension columns. Various kinds of samples were investigated and thus two different elution conditions were needed to cause full elution from the first dimension columns. We compared LC × LC chromatograms, contours plots, and fcoverage plots by measuring peak capacities, peak numbers, relative spatial coverage, correlation values, etc. The major finding of this study is that the carbon phase due to its rather different selectivity from other reversed phases is reasonably orthogonal to a variety of common types of bonded reversed phases. Thus quite surprisingly the six different first dimension stationary phases all showed generally similar separation patterns when paired to the second dimension carbon phase. This result greatly simplifies the task of choosing the correct pair of phases for RP × RP. PMID:21840009

Isolation of n-decyl-alpha(1-->6) isomaltoside from a technical APG mixture and its identification by the parallel use of LC-MS and NMR spectroscopy

PubMed

Billian; Hock; Doetzer; Stan; Dreher

2000-10-15

The identification of n-decyl alpha(1-->6)isomaltoside as a main component of technical alkyl polyglucoside (APG) mixtures by the parallel use of liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy is described. Following enrichment on a styrene-divinylbenzene-based solid-phase extraction material, unknown components were separated by reversed-phase liquid chromatography (LC). Chemical characterization was achieved by both mass spectrometry and NMR spectroscopy. It is demonstrated that the combination of LC-MS with various NMR techniques is very suitable for stereochemical assignment of unknown components in technical APG mixtures.

LC3 fluorescent puncta in autophagosomes or in protein aggregates can be distinguished by FRAP analysis in living cells

PubMed Central

Wang, Liang; Chen, Min; Yang, Jie; Zhang, Zhihong

2013-01-01

LC3 is a marker protein that is involved in the formation of autophagosomes and autolysosomes, which are usually characterized and monitored by fluorescence microscopy using fluorescent protein-tagged LC3 probes (FP-LC3). FP-LC3 and even endogenous LC3 can also be incorporated into intracellular protein aggregates in an autophagy-independent manner. However, the dynamic process of LC3 associated with autophagosomes and autolysosomes or protein aggregates in living cells remains unclear. Here, we explored the dynamic properties of the two types of FP-LC3-containing puncta using fluorescence microscopy techniques, including fluorescence recovery after photobleaching (FRAP) and fluorescence resonance energy transfer (FRET). The FRAP data revealed that the fluorescent signals of FP-LC3 attached to phagophores or in mature autolysosomes showed either minimal or no recovery after photobleaching, indicating that the dissociation of LC3 from the autophagosome membranes may be very slow. In contrast, FP-LC3 in the protein aggregates exhibited nearly complete recovery (more than 80%) and rapid kinetics of association and dissociation (half-time < 1 sec), indicating a rapid exchange occurs between the aggregates and cytoplasmic pool, which is mainly due to the transient interaction of LC3 and SQSTM1/p62. Based on the distinct dynamic properties of FP-LC3 in the two types of punctate structures, we provide a convenient and useful FRAP approach to distinguish autophagosomes from LC3-involved protein aggregates in living cells. Using this approach, we find the FP-LC3 puncta that adjacently localized to the phagophore marker ATG16L1 were protein aggregate-associated LC3 puncta, which exhibited different kinetics compared with that of autophagic structures. PMID:23482084

Imaging protein complex formation in the autophagy pathway: analysis of the interaction of LC3 and Atg4BC74A in live cells using Förster resonance energy transfer and fluorescence recovery after photobleaching

NASA Astrophysics Data System (ADS)

Kraft, Lewis J.; Kenworthy, Anne K.

2012-01-01

The protein microtubule-associated protein 1, light chain 3 (LC3) functions in autophagosome formation and plays a central role in the autophagy pathway. Previously, we found LC3 diffuses more slowly in cells than is expected for a freely diffusing monomer, suggesting it may constitutively associate with a macromolecular complex containing other protein components of the pathway. In the current study, we used Förster resonance energy transfer (FRET) microscopy and fluorescence recovery after photobleaching (FRAP) to investigate the interactions of LC3 with Atg4BC74A, a catalytically inactive mutant of the cysteine protease involved in lipidation and de-lipidation of LC3, as a model system to probe protein complex formation in the autophagy pathway. We show Atg4BC74A is in FRET proximity with LC3 in both the cytoplasm and nucleus of living cells, consistent with previous biochemical evidence that suggests these proteins directly interact. In addition, overexpressed Atg4BC74A diffuses significantly more slowly than predicted based on its molecular weight, and its translational diffusion coefficient is significantly slowed upon coexpression with LC3 to match that of LC3 itself. Taken together, these results suggest Atg4BC74A and LC3 are contained within the same multiprotein complex and that this complex exists in both the cytoplasm and nucleoplasm of living cells.

Identification of Forced Degradation Products of Itopride by LC-PDA and LC-MS.

PubMed

Joshi, Payal; Bhoir, Suvarna; Bhagwat, A M; Vishwanath, K; Jadhav, R K

2011-05-01

Degradation products of itopride formed under different forced conditions have been identified using LC-PDA and LC-MS techniques. Itopride was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, dry and wet heat, in accordance with the International Conference on Harmonization. The stress solutions were chromatographed on reversed phase C18 (250×4.6 mm, 5 μm) column with a mobile phase methanol:water (55:45, v/v) at a detection wavelength of 215 nm. Itopride degraded in acid, alkali and oxidative stress conditions. The stability indicating method was developed and validated. The degradation pathway of the drug to products II-VIII is proposed.

Identification of Forced Degradation Products of Itopride by LC-PDA and LC-MS

PubMed Central

Joshi, Payal; Bhoir, Suvarna; Bhagwat, A. M.; Vishwanath, K.; Jadhav, R. K.

2011-01-01

Degradation products of itopride formed under different forced conditions have been identified using LC-PDA and LC-MS techniques. Itopride was subjected to forced degradation under the conditions of hydrolysis, photolysis, oxidation, dry and wet heat, in accordance with the International Conference on Harmonization. The stress solutions were chromatographed on reversed phase C18 (250×4.6 mm, 5 μm) column with a mobile phase methanol:water (55:45, v/v) at a detection wavelength of 215 nm. Itopride degraded in acid, alkali and oxidative stress conditions. The stability indicating method was developed and validated. The degradation pathway of the drug to products II-VIII is proposed. PMID:22457552

Evaluation of the immunomodulatory effect of the 14 kDa protein isolated from aged garlic extract on dendritic cells.

PubMed

Ahmadabad, Hasan Namdar; Hassan, Zuhair Mohammad; Safari, Elahe; Bozorgmehr, Mahmood; Ghazanfari, Tooba; Moazzeni, Seyed Mohammad

2011-01-01

Garlic is used all over the world for treatment of different diseases. A wide range of biological activities of garlic has been verified in vitro and in vivo. One of major proteins of garlic which has been isolated and purified is the 14 kDa protein. This protein has been shown to have immunomodulatory effects. In this study, the effect of the 14 kDa protein isolated from aged garlic extract (AGE) was investigated on maturation and immunomodulatory activity of dendritic cells (DC). Proteins were purified from AGE by biochemical method; the semi-purified 14 kDa protein was run on gel filtration Sephadex G50 and its purity was checked by SDS-PAGE. DC were isolated from spleen of BALB/c mice by Nycodenz centrifugation and their adhesiveness to plastic dish. 14 kDa protein from AGE was added to overnight culture of DC medium and the expression percentage of CD40, CD86, and MHC-II was evaluated by flowcytometric analysis. Also, proliferation of T-cells was measured by allogenic mixed lymphocyte reaction (MLR) test. The purified 14 kDa protein isolated from AGE increased the expression of CD40 molecule on DC, but it did not influence CD86 and MHCII molecules. Furthermore, no significant differences were noticed in the pulsed-DC with 14 kDa protein and non-pulsed DC on the MLR. Copyright © 2011 Elsevier Inc. All rights reserved.

SU(1,1)-type light-atom-correlated interferometer

NASA Astrophysics Data System (ADS)

Ma, Hongmei; Li, Dong; Yuan, Chun-Hua; Chen, L. Q.; Ou, Z. Y.; Zhang, Weiping

2015-08-01

The quantum correlation of light and atomic collective excitation can be used to compose an SU(1,1)-type hybrid light-atom interferometer, where one arm in the optical SU(1,1) interferometer is replaced by the atomic collective excitation. The phase-sensing probes include not only the photon field but also the atomic collective excitation inside the interferometer. For a coherent squeezed state as the phase-sensing field, the phase sensitivity can approach the Heisenberg limit under the optimal conditions. We also study the effects of the loss of light field and the dephasing of atomic excitation on the phase sensitivity. This kind of active SU(1,1) interferometer can also be realized in other systems, such as circuit quantum electrodynamics in microwave systems, which provides a different method for basic measurement using the hybrid interferometers.

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

PubMed

Sugimoto, H; Yamashita, S

1999-05-18

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0

An outer arm dynein light chain acts in a conformational switch for flagellar motility

PubMed Central

Patel-King, Ramila S.

2009-01-01

A system distinct from the central pair–radial spoke complex was proposed to control outer arm dynein function in response to alterations in the mechanical state of the flagellum. In this study, we examine the role of a Chlamydomonas reinhardtii outer arm dynein light chain that associates with the motor domain of the γ heavy chain (HC). We demonstrate that expression of mutant forms of LC1 yield dominant-negative effects on swimming velocity, as the flagella continually beat out of phase and stall near or at the power/recovery stroke switchpoint. Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme. Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain. We propose that this γ HC–LC1–microtubule ternary complex functions as a conformational switch to control outer arm activity. PMID:19620633

Glycoproteins Enrichment and LC-MS/MS Glycoproteomics in Central Nervous System Applications.

PubMed

Zhu, Rui; Song, Ehwang; Hussein, Ahmed; Kobeissy, Firas H; Mechref, Yehia

2017-01-01

Proteins and glycoproteins play important biological roles in central nervous systems (CNS). Qualitative and quantitative evaluation of proteins and glycoproteins expression in CNS is critical to reveal the inherent biomolecular mechanism of CNS diseases. This chapter describes proteomic and glycoproteomic approaches based on liquid chromatography/tandem mass spectrometry (LC-MS or LC-MS/MS) for the qualitative and quantitative assessment of proteins and glycoproteins expressed in CNS. Proteins and glycoproteins, extracted by a mass spectrometry friendly surfactant from CNS samples, were subjected to enzymatic (tryptic) digestion and three down-stream analyses: (1) a nano LC system coupled with a high-resolution MS instrument to achieve qualitative proteomic profile, (2) a nano LC system combined with a triple quadrupole MS to quantify identified proteins, and (3) glycoprotein enrichment prior to LC-MS/MS analysis. Enrichment techniques can be applied to improve coverage of low abundant glycopeptides/glycoproteins. An example described in this chapter is hydrophilic interaction liquid chromatographic (HILIC) enrichment to capture glycopeptides, allowing efficient removal of peptides. The combination of three LC-MS/MS-based approaches is capable of the investigation of large-scale proteins and glycoproteins from CNS with an in-depth coverage, thus offering a full view of proteins and glycoproteins changes in CNS.

LC-IMS-MS Feature Finder

DOE Office of Scientific and Technical Information (OSTI.GOV)

2013-03-07

LC-IMS-MS Feature Finder is a command line software application which searches for possible molecular ion signatures in multidimensional liquid chromatography, ion mobility spectrometry, and mass spectrometry data by clustering deisotoped peaks with similar monoisotopic mass values, charge states, elution times, and drift times. The software application includes an algorithm for detecting multiple conformations and co-eluting species in the ion mobility dimension. LC-IMS-MS Feature Finder is designed to create an output file with detected features that includes associated information about the detected features.

Towards simulating and quantifying the light-cone EoR 21-cm signal

NASA Astrophysics Data System (ADS)

Mondal, Rajesh; Bharadwaj, Somnath; Datta, Kanan K.

2018-02-01

The light-cone (LC) effect causes the Epoch of Reionization (EoR) 21-cm signal T_b (\\hat{n}, ν ) to evolve significantly along the line-of-sight (LoS) direction ν. In the first part of this paper, we present a method to properly incorporate the LC effect in simulations of the EoR 21-cm signal that includes peculiar velocities. Subsequently, we discuss how to quantify the second-order statistics of the EoR 21-cm signal in the presence of the LC effect. We demonstrate that the 3D power spectrum P(k) fails to quantify the entire information because it assumes the signal to be ergodic and periodic, whereas the LC effect breaks these conditions along the LoS. Considering a LC simulation centred at redshift 8 where the mean neutral fraction drops from 0.65 to 0.35 across the box, we find that P(k) misses out ˜ 40 per cent of the information at the two ends of the 17.41 MHz simulation bandwidth. The multifrequency angular power spectrum (MAPS) C_{ℓ}(ν_1,ν_2) quantifies the statistical properties of T_b (\\hat{n}, ν ) without assuming the signal to be ergodic and periodic along the LoS. We expect this to quantify the entire statistical information of the EoR 21-cm signal. We apply MAPS to our LC simulation and present preliminary results for the EoR 21-cm signal.

Light deprivation damages monoamine neurons and produces a depressive behavioral phenotype in rats

PubMed Central

Gonzalez, M. M. C.; Aston-Jones, G.

2008-01-01

Light is an important environmental factor for regulation of mood. There is a high frequency of seasonal affective disorder in high latitudes where light exposure is limited, and bright light therapy is a successful antidepressant treatment. We recently showed that rats kept for 6 weeks in constant darkness (DD) have anatomical and behavioral features similar to depressed patients, including dysregulation of circadian sleep–waking rhythms and impairment of the noradrenergic (NA)-locus coeruleus (LC) system. Here, we analyzed the cell viability of neural systems related to the pathophysiology of depression after DD, including NA-LC, serotoninergic-raphe nuclei and dopaminergic-ventral tegmental area neurons, and evaluated the depressive behavioral profile of light-deprived rats. We found increased apoptosis in the three aminergic systems analyzed when compared with animals maintained for 6 weeks in 12:12 light-dark conditions. The most apoptosis was observed in NA-LC neurons, associated with a significant decrease in the number of cortical NA boutons. Behaviorally, DD induced a depression-like condition as measured by increased immobility in a forced swim test (FST). DD did not appear to be stressful (no effect on adrenal or body weights) but may have sensitized responses to subsequent stressors (increased fecal number during the FST). We also found that the antidepressant desipramine decreases these neural and behavioral effects of light deprivation. These findings indicate that DD induces neural damage in monoamine brain systems and this damage is associated with a depressive behavioral phenotype. Our results suggest a mechanism whereby prolonged limited light intensity could negatively impact mood. PMID:18347342

Preprocessing and Analysis of LC-MS-Based Proteomic Data

PubMed Central

Tsai, Tsung-Heng; Wang, Minkun; Ressom, Habtom W.

2016-01-01

Liquid chromatography coupled with mass spectrometry (LC-MS) has been widely used for profiling protein expression levels. This chapter is focused on LC-MS data preprocessing, which is a crucial step in the analysis of LC-MS based proteomics. We provide a high-level overview, highlight associated challenges, and present a step-by-step example for analysis of data from LC-MS based untargeted proteomic study. Furthermore, key procedures and relevant issues with the subsequent analysis by multiple reaction monitoring (MRM) are discussed. PMID:26519169

Silver ion chromatography for peak resolution enhancement: Application to the preparative separation of two sesquiterpenes using online heart-cutting LC-LC technique.

PubMed

Yang, Yang; Zhang, Yongmin; Wei, Chong; Li, Jing; Sun, Wenji

2018-09-01

Silver ion chromatography, utilizing columns packed with silver ions bonded to silica gel, has proved to be an invaluable technique for the analysis of some positional isomers. In this work, silver ion chromatography by combination with online heart-cutting LC-LC technique for the preparative separation of two sesquiterpenes positional isomers from a natural product was investigated. On the basis of the evaluation that silver ion content impacts on the separation, the laboratory-made silver ion columns, utilizing silica gel impregnated with 15% silver nitrate as column packing materials, were used for peak resolution improvement of these two isomers and the preparative separation of them in heart-cutting LC-LC. The relationship among the maximal sample load, flow rate and peak resolution in the silver ion column were optimized, and the performance of the silver ion column was compared with conventional C 18 column and silica gel column. Based on the developed chromatographic conditions, online heart-cutting LC-LC chromatographic separation system in combination with a silica gel column and a silver ion column that was applied to preparative separation of these two isomers from a traditional Chinese medicine, Inula racemosa Hook.f., was established. The results showed that the online heart-cutting LC-LC technique by combination of a silica gel column and a silver ion column for the preparative separation of these two positional isomers from this natural plant was superior to the preparative separation performed on a single-column system with C 18 column or silica gel column. Copyright © 2018 Elsevier B.V. All rights reserved.

The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments.

PubMed

Cannataro, Mario; Cuda, Giovanni; Gaspari, Marco; Greco, Sergio; Tradigo, Giuseppe; Veltri, Pierangelo

2007-07-15

Isotope-coded affinity tags (ICAT) is a method for quantitative proteomics based on differential isotopic labeling, sample digestion and mass spectrometry (MS). The method allows the identification and relative quantification of proteins present in two samples and consists of the following phases. First, cysteine residues are either labeled using the ICAT Light or ICAT Heavy reagent (having identical chemical properties but different masses). Then, after whole sample digestion, the labeled peptides are captured selectively using the biotin tag contained in both ICAT reagents. Finally, the simplified peptide mixture is analyzed by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nevertheless, the ICAT LC-MS/MS method still suffers from insufficient sample-to-sample reproducibility on peptide identification. In particular, the number and the type of peptides identified in different experiments can vary considerably and, thus, the statistical (comparative) analysis of sample sets is very challenging. Low information overlap at the peptide and, consequently, at the protein level, is very detrimental in situations where the number of samples to be analyzed is high. We designed a method for improving the data processing and peptide identification in sample sets subjected to ICAT labeling and LC-MS/MS analysis, based on cross validating MS/MS results. Such a method has been implemented in a tool, called EIPeptiDi, which boosts the ICAT data analysis software improving peptide identification throughout the input data set. Heavy/Light (H/L) pairs quantified but not identified by the MS/MS routine, are assigned to peptide sequences identified in other samples, by using similarity criteria based on chromatographic retention time and Heavy/Light mass attributes. EIPeptiDi significantly improves the number of identified peptides per sample, proving that the proposed method has a considerable impact on the protein identification process and, consequently, on

Fast-responding liquid crystal light-valve technology for color-sequential display applications

NASA Astrophysics Data System (ADS)

Janssen, Peter J.; Konovalov, Victor A.; Muravski, Anatoli A.; Yakovenko, Sergei Y.

1996-04-01

A color sequential projection system has some distinct advantages over conventional systems which make it uniquely suitable for consumer TV as well as high performance professional applications such as computer monitors and electronic cinema. A fast responding light-valve is, clearly, essential for a good performing system. Response speed of transmissive LC lightvalves has been marginal thus far for good color rendition. Recently, Sevchenko Institute has made some very fast reflective LC cells which were evaluated at Philips Labs. These devices showed sub millisecond-large signal-response times, even at room temperature, and produced good color in a projector emulation testbed. In our presentation we describe our highly efficient color sequential projector and demonstrate its operation on video tape. Next we discuss light-valve requirements and reflective light-valve test results.

Detection of N-(1-deoxy-D-fructos-1-yl) Fumonisins B₂ and B₃ in Corn by High-Resolution LC-Orbitrap MS.

PubMed

Matsuo, Yosuke; Takahara, Kentaro; Sago, Yuki; Kushiro, Masayo; Nagashima, Hitoshi; Nakagawa, Hiroyuki

2015-09-16

The existence of glucose conjugates of fumonisin B₂ (FB₂) and fumonisin B₃ (FB₃) in corn powder was confirmed for the first time. These "bound-fumonisins" (FB₂ and FB₃ bound to glucose) were identified as N-(1-deoxy-D-fructos-1-yl) fumonisin B₂ (NDfrc-FB₂) and N-(1-deoxy-D-fructos-1-yl) fumonisin B₃ (NDfrc-FB₃) respectively, based on the accurate mass measurements of characteristic ions and fragmentation patterns using high-resolution liquid chromatography-Orbitrap mass spectrometry (LC-Orbitrap MS) analysis. Treatment on NDfrc-FB₂ and NDfrc-FB₃ with the o-phthalaldehyde (OPA) reagent also supported that D-glucose binding to FB₂ and FB₃ molecules occurred to their primary amine residues.

LC-MS metabolic profiling of Arabidopsis thaliana plant leaves and cell cultures: optimization of pre-LC-MS procedure parameters.

PubMed

t'Kindt, Ruben; De Veylder, Lieven; Storme, Michael; Deforce, Dieter; Van Bocxlaer, Jan

2008-08-01

This study treats the optimization of methods for homogenizing Arabidopsis thaliana plant leaves as well as cell cultures, and extracting their metabolites for metabolomics analysis by conventional liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS). Absolute recovery, process efficiency and procedure repeatability have been compared between different pre-LC-MS homogenization/extraction procedures through the use of samples fortified before extraction with a range of representative metabolites. Hereby, the magnitude of the matrix effect observed in the ensuing LC-MS based metabolomics analysis was evaluated. Based on relative recovery and repeatability of key metabolites, comprehensiveness of extraction (number of m/z-retention time pairs) and clean-up potential of the approach (minimum matrix effects), the most appropriate sample pre-treatment was adopted. It combines liquid nitrogen homogenization for plant leaves with thermomixer based extraction using MeOH/H(2)O 80/20. As such, an efficient and highly reproducible LC-MS plant metabolomics set-up is achieved, as illustrated by the obtained results for both LC-MS (8.88%+/-5.16 versus 7.05%+/-4.45) and technical variability (12.53%+/-11.21 versus 9.31%+/-6.65) data in a comparative investigation of A. thaliana plant leaves and cell cultures, respectively.

Light-Driven Chiral Molecular Motors for Passive Agile Filters

DTIC Science & Technology

2014-05-20

liquid crystal , we fabricated the self-organized, phototubable 3D photonic superstructure, i.e. photoresponsive monodisperse cholesteric liquid...systems for applications. Here the new light-driven chiral molecular switch and upconversion nanoparticles, doped in a liquid crystal media, were...the bottom-up nanofabrication of intelligent molecular devices. Light-driven chiral molecular switches or motors in liquid crystal (LC) media that

Induction of hsp70 by the herbicide oxyfluorfen (Goal) in the Egyptian Nile fish, Oreochromis niloticus.

PubMed

Hassanein, H M; Banhawy, M A; Soliman, F M; Abdel-Rehim, S A; Müller, W E; Schröder, H C

1999-07-01

This paper deals with the expression of the biomarker hsp70 in the liver and kidney of the freshwater fish Oreochromis niloticus following exposure to the herbicide oxyfluorfen (Goal). Fishes were exposed to three concentrations, the 96-h LC50 (3 mg/L), the 96-h (1/2)LC50 (1.5 mg/L), and the 96-h (1/4)LC50 (0.75 mg/L) of oxyfluorfen for 6, 15, and 24 days, respectively, and samples were taken at three different time periods for each concentration. The livers responded to the herbicide by an induction of the expression of both the constitutive (hsp75; Mr 75 kDa) and the inducible (hsp73; Mr 73 kDa) hsp70 proteins. In kidney, the herbicide induced a time-dependent increase in the expression of the constitutive hsp70 (hsp75) as well, but the inducible hsp70 (hsp73) required much longer incubation periods to reach maximal levels (15 and 24 days). Our results suggest that expression of hsp70 in fish is a sensitive indicator of cellular responses to herbicide exposure in the aquatic environment.

Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins.

PubMed

Johansen, T; Birgisdottir, Å B; Huber, J; Kniss, A; Dötsch, V; Kirkin, V; Rogov, V V

2017-01-01

LC3/GABARAP proteins (LC3/GABARAPs) are mammalian orthologues of yeast Atg8, small ubiquitin (Ub)-like proteins (UBLs) whose covalent attachment to lipid membranes is crucial for the growth and closure of the double membrane vesicle called the autophagosome. In the past decade, it was demonstrated that Atg8/LC3/GABARAPs are also required for autophagic degradation of cargos in a selective fashion. Cargo selectivity is ensured by receptor proteins, such as p62/SQSTM1, NBR1, Cue5, Atg19, NIX, Atg32, NCOA4, and FAM134B, which simultaneously bind Atg8/LC3/GABARAPs and the cargo together, thereby linking the core autophagic machinery to the target structure: a protein, an organelle, or a pathogen. LC3-interacting regions (LIRs) are short linear motifs within selective autophagy receptors and some other structural and signaling proteins (e.g., ULK1, ATG13, FIP200, and Dvl2), which mediate binding to Atg8/LC3/GABARAPs. Identification and characterization of LIR-containing proteins have provided important insights into the biology of the autophagy pathway, and studying their interactions with the core autophagy machinery represents a growing area of autophagy research. Here, we present protocols for the identification of LIR-containing proteins, i.e., by yeast-two-hybrid screening, glutathione S-transferase (GST) pulldown experiments, and peptide arrays. The use of two-dimensional peptide arrays also represents a powerful method to identify the residues of the LIR motif that are critical for binding. We also describe a biophysical method for studying interactions between Atg8/LC3/GABARAP and LIR-containing proteins and a protocol for preparation and purification of LIR peptides. © 2017 Elsevier Inc. All rights reserved.

A 92-kDa human immunostimulatory protein.

PubMed Central

Fontan, E; Briend, E; Saklani-Jusforgues, H; d'Alayer, J; Vandekerckhove, J; Fauve, R M

1994-01-01

We purified to apparent homogeneity a human urinary glycoprotein of 92 kDa (HGP.92) that, administered intravenously at 250 micrograms/kg, fully protected mice against a lethal inoculum of Listeria monocytogenes. Since HGP.92 protected scid mice, which lack B and T lymphocytes, this increased resistance to Listeria did not appear to be lymphocyte mediated. Furthermore, inflammatory macrophages incubated with 6 nM HGP.92 inhibited the growth of Lewis carcinoma cells in vitro. These two activities appeared to depend on an oligosaccharide moiety, as they were lost after N-Glycanase treatment of HGP.92. Thus, the biological activity of HGP.92 was in some way related to a glycan moiety. Images PMID:8078887

An FD-LC-MS/MS Proteomic Strategy for Revealing Cellular Protein Networks: A Conditional Superoxide Dismutase 1 Knockout Cells

PubMed Central

Ichibangase, Tomoko; Sugawara, Yasuhiro; Yamabe, Akio; Koshiyama, Akiyo; Yoshimura, Akari; Enomoto, Takemi; Imai, Kazuhiro

2012-01-01

Systems biology aims to understand biological phenomena in terms of complex biological and molecular interactions, and thus proteomics plays an important role in elucidating protein networks. However, many proteomic methods have suffered from their high variability, resulting in only showing altered protein names. Here, we propose a strategy for elucidating cellular protein networks based on an FD-LC-MS/MS proteomic method. The strategy permits reproducible relative quantitation of differences in protein levels between different cell populations and allows for integration of the data with those obtained through other methods. We demonstrate the validity of the approach through a comparison of differential protein expression in normal and conditional superoxide dismutase 1 gene knockout cells and believe that beginning with an FD-LC-MS/MS proteomic approach will enable researchers to elucidate protein networks more easily and comprehensively. PMID:23029042

Crystal Structure of the 25 kDa Subunit of Human Cleavage Factor I{m}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coseno,M.; Martin, G.; Berger, C.

Cleavage factor Im is an essential component of the pre-messenger RNA 3'-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein-protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic {alpha}/{beta}/{alpha} fold and a conserved catalytic sequence or Nudix box. We present heremore » the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Angstroms, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3'-end processing.« less

Anti-nociceptive effect of a conjugate of substance P and light chain of botulinum neurotoxin type A.

PubMed

Mustafa, Golam; Anderson, Ethan M; Bokrand-Donatelli, Yvonne; Neubert, John K; Caudle, Robert M

2013-11-01

Neuropathic pain is a debilitating condition resulting from damage to sensory transmission pathways in the peripheral and central nervous system. A potential new way of treating chronic neuropathic pain is to target specific pain-processing neurons based on their expression of particular receptor molecules. We hypothesized that a toxin-neuropeptide conjugate would alter pain by first being taken up by specific receptors for the neuropeptide expressed on the neuronal cells. Then, once inside the cell the toxin would inhibit the neurons' activity without killing the neurons, thereby providing pain relief without lesioning the nervous system. In an effort to inactivate the nociceptive neurons in the trigeminal nucleus caudalis in mice, we targeted the NK1 receptor (NK1R) using substance P (SP). The catalytically active light chain of botulinum neurotoxin type A (LC/A) was conjugated with SP. Our results indicate that the conjugate BoNT/A-LC:SP is internalized in cultured NK1R-expressing neurons and also cleaves the target of botulinum toxin, a component-docking motif necessary for release of neurotransmitters called SNAP-25. The conjugate was next tested in a murine model of Taxol-induced neuropathic pain. An intracisternal injection of BoNT/A-LC:SP decreased thermal hyperalgesia as measured by the operant orofacial nociception assay. These findings indicate that conjugates of the light chain of botulinum toxin are extremely promising agents for use in suppressing neuronal activity for extended time periods, and that BoNT/A-LC:SP may be a useful agent for treating chronic pain. Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Draft Genome Sequences of Two Bacillus thuringiensis Strains and Characterization of a Putative 41.9-kDa Insecticidal Toxin

PubMed Central

Palma, Leopoldo; Muñoz, Delia; Berry, Colin; Murillo, Jesús; Caballero, Primitivo

2014-01-01

In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 µg/cm2 and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein’s target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins. PMID:24784323

Crystallization and preliminary crystallographic analysis of human Atg4B–LC3 complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Satoo, Kenji; Suzuki, Nobuo N.; Fujioka, Yuko

2007-02-01

Human Atg4B and LC3 were expressed, purified and crystallized as a complex. Diffraction data were collected to a resolution of 1.9 Å. The reversible modification of Atg8 with phosphatidylethanolamine (PE) is crucial for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Atg4 is a cysteine protease that is responsible for the processing and deconjugation of Atg8. Human Atg4B (HsAtg4B; a mammalian orthologue of yeast Atg4) and LC3 (a mammalian orthologue of yeast Atg8) were expressed and purified and two complexes, one consisting of HsAtg4B(1–354) and LC3(1–120) (complex I; the product complex) and the other consisting ofmore » HsAtg4B(1–354) and LC3(1–124) (complex II; the substrate complex), were crystallized using polyethylene glycol 3350 as a precipitant. In both complexes His280 of HsAtg4B was mutated to alanine. The crystals belong to the same space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 47.5, b = 91.8, c = 102.6 Å for complex I and a = 46.9, b = 90.9, c = 102.5 Å for complex II. Diffraction data were collected to a resolution of 1.9 Å from both crystals.« less

Calibration of a spatial light modulator containing dual frequency liquid crystal

NASA Astrophysics Data System (ADS)

Gu, Dong-Feng; Winker, Bruce; Wen, Bing; Taber, Don; Brackley, Andrew; Wirth, Allan; Albanese, Marc; Landers, Frank

2005-08-01

Characterization and calibration process for a liquid crystal (LC) spatial light modulator (SLM) containing dual frequency liquid crystal is described. Special care was taken when dealing with LC cell gap non-uniformity and defect pixels. The calibration results were fed into a closed loop control algorithm to demonstrate correction of wavefront distortions. The performance characteristics of the device were reported. Substantial improvements were made in speed (bandwidth), resolution, power consumption and system weight/volume.

Tunable properties of light propagation in photonic liquid crystal fibers

NASA Astrophysics Data System (ADS)

Szaniawska, K.; Nasilowski, T.; Woliński, T. R.; Thienpont, H.

2006-12-01

Tunable properties of light propagation in photonic crystal fibers filled with liquid crystals, called photonic liquid crystal fibers (PLCFs) are presented. The propagation properties of PLCFs strongly depend on contrast between refractive indices of the solid core (pure silica glass) and liquid crystals (LCs) filing the holes of the fiber. Due to relatively strong thermo-optical effect, we can change the refractive index of the LC by changing its temperature. Numerical analysis of light propagation in PLCF, based on two simulation methods, such as finite difference (FD) and multipole method (MM) is presented. The numerical results obtained are in good agreement with our earlier experimental results presented elsewhere [1].

The regulation of smooth muscle contractility by zipper-interacting protein kinase.

PubMed

Ihara, Eikichi; MacDonald, Justin A

2007-01-01

Smooth muscle contractility is mainly regulated by phosphorylation of the 20 kDa myosin light chains (LC20), a process that is controlled by the opposing activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). Recently, intensive research has revealed that various protein kinase networks including Rho-kinase, integrin-linked kinase, zipper-interacting protein kinase (ZIPK), and protein kinase C (PKC) are involved in the regulation of LC20 phosphorylation and have important roles in modulating smooth muscle contractile responses to Ca2+ (i.e., Ca2+ sensitization and Ca2+ desensitization). Here, we review the general background and structure of ZIPK and summarize our current understanding of its involvement in a number of cell processes including cell death (apoptosis), cell motility, and smooth muscle contraction. ZIPK has been found to induce the diphosphorylation of LC20 at Ser-19 and Thr-18 in a Ca2+-independent manner and to regulate MLCP activity directly through its phosphorylation of the myosin-targeting subunit of MLCP or indirectly through its phosphorylation of the PKC-potentiated inhibitory protein of MLCP. Future investigations of ZIPK function in smooth muscle will undoubtably focus on determining the mechanisms that regulate its cellular activity, including the identification of upstream signaling pathways, the characterization of autoinhibitory domains and regulatory phosphorylation sites, and the development of specific inhibitor compounds.

p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy.

PubMed

Pankiv, Serhiy; Clausen, Terje Høyvarde; Lamark, Trond; Brech, Andreas; Bruun, Jack-Ansgar; Outzen, Heidi; Øvervatn, Aud; Bjørkøy, Geir; Johansen, Terje

2007-08-17

Protein degradation by basal constitutive autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitin-binding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related gamma-aminobutyrate receptor-associated protein and gamma-aminobutyrate receptor-associated-like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62- and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62- and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 microm diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.

Determination of toxins involved in ciguatera fish poisoning in the Pacific by LC/MS.

PubMed

Yogi, Kentaro; Sakugawa, Satsuki; Oshiro, Naomasa; Ikehara, Tsuyoshi; Sugiyama, Kiminori; Yasumoto, Takeshi

2014-01-01

Ciguatera fish poisoning is the most extensive and difficult to control of the seafood poisonings. To facilitate monitoring of fish toxicity, toxin profiles were investigated by an LC/MS/MS method using 14 reference toxins on eight representative species of fish collected in four different areas of the Pacific. Snappers and groupers from Okinawa contained ciguatoxin-1B (CTX1B) and two deoxy congeners at variable but species-specific ratios, while red snapper, Lutjanus bohar, from Minamitorishima, and amberjack, Seriola dumerili, from Hawaii, contained both CTX1B-type and CTX3C-type toxins. Spotted knifejaw, Oplegnathus punctatus, from Okinawan waters, contained mainly CTX4A and CTX4B, but the same species caught at Miyazaki was contaminated primarily with the CTX3C-type toxins. Otherwise, the toxin profiles were consistently species-specific in fish collected from various locations around Okinawa over 20 years. The LC/MS/MS and mouse bioassay results agreed well, indicating the LC/MS/MS method is a promising alternative to the mouse bioassay. Pure CTX1B and CTX3C were prepared for use in future LC/MS/MS analysis.

Simultaneous Quantitation of Atenolol, Metoprolol, and Propranolol in Biological Matrices Via LC/MS

DTIC Science & Technology

2005-05-01

Simultaneous Quantitation of Atenolol, Metoprolol , and Propranolol in Biological Matrices Via LC/MS Robert D. Johnson Russell J. Lewis Civil...authorized 1 SIMULTANEOUS QUANTITATION OF ATENOLOL, METOPROLOL , AND PROPRANOLOL IN BIOLOGICAL MATRICES VIA LC/MS INTRODUCTION The Federal Aviation...detect beta-blocker compounds such as atenolol, metoprolol , or propranolol in the submitted biological samples. In forensic toxicol- ogy laboratories

Indocyanine Green-Loaded Liposomes for Light-Triggered Drug Release.

PubMed

Lajunen, Tatu; Kontturi, Leena-Stiina; Viitala, Lauri; Manna, Moutusi; Cramariuc, Oana; Róg, Tomasz; Bunker, Alex; Laaksonen, Timo; Viitala, Tapani; Murtomäki, Lasse; Urtti, Arto

2016-06-06

Light-triggered drug delivery systems enable site-specific and time-controlled drug release. In previous work, we have achieved this with liposomes containing gold nanoparticles in the aqueous core. Gold nanoparticles absorb near-infrared light and release the energy as heat that increases the permeability of the liposomal bilayer, thus releasing the contents of the liposome. In this work, we replaced the gold nanoparticles with the clinically approved imaging agent indocyanine green (ICG). The ICG liposomes were stable at storage conditions (4-22 °C) and at body temperature, and fast near-infrared (IR) light-triggered drug release was achieved with optimized phospholipid composition and a 1:50 ICG-to-lipid molar ratio. Encapsulated small molecular calcein and FITC-dextran (up to 20 kDa) were completely released from the liposomes after light exposure for 15 s. Location of ICG in the PEG layer of the liposomes was simulated with molecular dynamics. ICG has important benefits as a light-triggering agent in liposomes: fast content release, improved stability, improved possibility of liposomal size control, regulatory approval to use in humans, and the possibility of imaging the in vivo location of the liposomes based on the fluorescence of ICG. Near-infrared light used as a triggering mechanism has good tissue penetration and safety. Thus, ICG liposomes are an attractive option for light-controlled and efficient delivery of small and large drug molecules.

LC and ferromagnetic resonance in soft/hard magnetic microwires

NASA Astrophysics Data System (ADS)

Tian, Bin; Vazquez, Manuel

2015-12-01

The magnetic behavior of soft/hard biphase microwires is introduced here. The microwires consist of a Co59.1Fe14.8Si10.2B15.9 soft magnetic nucleus and a Co90Ni10 hard outer shell separated by an intermediate insulating Pyrex glass microtube. By comparing the resistance spectrums of welding the ends of metallic core (CC) or welding the metallic core and outer shell (CS) to the connector, it is found that one of the two peaks in the resistance spectrum is because the LC resonance depends on the inductor and capacitors in which one is the capacitor between the metallic core and outer shell, and the other is between the outer shell and connector. Correspondingly, another peak is for the ferromagnetic resonance of metallic core. After changing the capacitance of the capacitors, the frequency of LC resonance moves to high frequency band, and furthermore, the peak of LC resonance in the resistance spectrum disappeared. These magnetostatically coupled biphase systems are thought to be of large potential interest as sensing elements in sensor devices.

Conventional light chains inhibit the autonomous signaling capacity of the B cell receptor.

PubMed

Meixlsperger, Sonja; Köhler, Fabian; Wossning, Thomas; Reppel, Michael; Müschen, Markus; Jumaa, Hassan

2007-03-01

Signals from the B cell antigen receptor (BCR), consisting of mu heavy chain (muHC) and conventional light chain (LC), and its precursor the pre-BCR, consisting of muHC and surrogate light chain (SLC), via the adaptor protein SLP-65 regulate the development and function of B cells. Here, we compare the effect of SLC and conventional LC expression on receptor-induced Ca(2+) flux in B cells expressing an inducible form of SLP-65. We found that SLC expression strongly enhanced an autonomous ability of muHC to induce Ca(2+) flux irrespective of additional receptor crosslinking. In contrast, LC expression reduced this autonomous muHC ability and resulted in antigen-dependent Ca(2+) flux. These data indicate that autonomous ligand-independent signaling can be induced by receptor forms other than the pre-BCR. In addition, our data suggest that conventional LCs play an important role in the inhibition of autonomous receptor signaling, thereby allowing further B cell differentiation.

The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Graham S.T.; Seymour, Lauren V.; Boggs, Joan M.

2012-06-15

Highlights: Black-Right-Pointing-Pointer Full-length 21.5-kDa MBP isoform is translocated to the nucleus. Black-Right-Pointing-Pointer We hypothesized that the exon-II-encoded sequence contained the NLS. Black-Right-Pointing-Pointer We mutated this sequence in RFP-tagged constructs and transfected N19-cells. Black-Right-Pointing-Pointer Abolition of two key positively-charged residues resulted in loss of nuclear-trafficking. Black-Right-Pointing-Pointer The 21.5-kDa isoform of classic MBP contains a non-traditional PY-NLS. -- Abstract: The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca{sup 2+}-calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipidmore » membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two non-traditional PY-nuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.« less

Underivatized oxysterols and nanoLC-ESI-MS: A mismatch.

PubMed

Roberg-Larsen, Hanne; Vesterdal, Caroline; Wilson, Steven Ray; Lundanes, Elsa

2015-07-01

Due to their non-charged character, liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) measurements of oxysterols are often performed after derivatization with e.g. charged Girard reagents. However, derivatization reactions are time-consuming and may require numerous steps to remove excess reagent. In addition, extensive sample handling can be associated with cholesterol autoxidation, resulting in analyte artifacts and hence false positives. Nano scale liquid chromatography in combination with electrospray-mass spectrometry (nanoLC-ESI-MS) is a powerful tool for analyzing limited samples, due to substantially increased sensitivity compared to conventional LC-ESI-MS. The signal enhancement may compensate for the poor ionization of the oxysterols; hence we have explored the possibility to quantify oxysterols without derivatization using nanoLC-ESI-MS. Non-derivatized oxysterols and nanoLC were however not compatible, due to persistent and large carry-over. This was attributed to the extended contribution of surface to volume ratio in such miniaturized systems and interactions with the materials of the nanoLC instrumentation (e.g. adsorption to the fused silica tubing). Two contemporary MS instruments (Q-Exactive™ hybrid quadrupole-Orbitrap and TSQ Quantiva™ triple quadrupole) were used. However, both the MS and MS/MS spectra of non-derivatized oxysterols were ambiguous and/or unrepeatable for both of the instruments employed. Derivatizing oxysterols is more cumbersome, but provides more selective and reliable results, and Girard derivatization+nanoLC-ESI-MS continues to be our recommended choice for measuring oxysterols in very limited samples. These investigations also indicate that extra care should be taken to remove lipids prior to nanoLC of other analytes, as adsorbed oxysterols, etc. can compromise analysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Sunlight-switchable light shutter fabricated using liquid crystals doped with push-pull azobenzene.

PubMed

Oh, Seung-Won; Baek, Jong-Min; Yoon, Tae-Hoon

2016-11-14

We propose a sunlight-switchable light shutter using liquid crystal/polymer composite doped with push-pull azobenzene. The proposed light shutter is switchable between the translucent and transparent states by application of an electric field or by UV irradiation. Switching by UV irradiation is based on the change of the liquid crystal (LC) clearing point by the photo-isomerization effect of push-pull azobenzene. Under sunlight, the light shutter can be switched from the translucent to the transparent state by the nematic-isotropic phase transition of the LC domains triggered by trans-cis photo-isomerization of the push-pull azobenzene molecules. When the amount of sunlight is low because of cloud cover or when there is no sunlight at sunset, the light shutter rapidly relaxes from its transparent state back to its initial translucent state by the isotropic-nematic phase transition induced by cis-trans back-isomerization of the push-pull azobenzene molecules.

The purification and characterization of an 88-kDa Porphyromonas endodontalis 35406 protease.

PubMed

Rosen, G; Shoshani, M; Naor, R; Sela, M N

2001-12-01

A Porphyromonas endodontalis ATCC 35406 protease was purified from Triton X-114 cell extracts by preparative SDS-PAGE followed by electroelution. The purified enzyme exhibits a molecular size of 88 kDa and was dissociated into two polypeptides of 43 and 41 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The protease (pH optimum 7.5-8.0) degraded the extracellular matrix proteins fibrinogen and fibronectin. Collagen IV was also degraded at 37 degrees C but not at 28 degrees C. The protease also cleaved the bioactive peptide angiotensin at amino acid residue phenylalanine-8 and tyrosine-4 but failed to hydrolyze bradykinin, vasopressin and synthetic chromogenic substrates with phenylalanine or tyrosine at the P1 position. In addition, two peptidases were detected in P. endodontalis cells: a proline aminopeptidase that remained associated with the cell pellet after detergent extraction and peptidase/s that partitioned into the Triton X-114 phase after phase separation and degraded the bioactive peptides bradykinin and vasopressin. These P. endodontalis peptidases and proteases may play an important role in both the nutrition and pathogenicity of these assacharolytic microorganisms. The inactivation of bioactive peptides and degradation of extracellular matrix proteins by bacterial enzymes may contribute to the damage of host tissues accompanied with endodontic infections.

Expression of Clara cell 10-kDa protein and trefoil factor family 1 in patients with chronic rhinosinusitis and nasal polyps

PubMed Central

Wang, Yuanyuan; Wang, Zong-Feng; Zhang, Zhili; Su, Yi

2018-01-01

The current study measured the expression of Clara cell 10-kDa protein (CC10) and trefoil factor family 1 (TFF1) in the sinus mucosa of patients exhibiting chronic rhinosinusitis (CRS) and nasal polyps (NP). CC10 and TFF1 expression in the sinus mucosa of the control group and patients with CRS and NP was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting and immunohistochemistry. The correlation between CC10 and TFF1 expression was further analyzed using Spearman's correlation analysis. The expression of TFF1 was significantly increased in the sinus mucosa of patients with CRS and NP, whereas CC10 expression was significantly decreased compared with controls. Spearman's correlation analysis identified a negative correlation between CC10 and TFF1 expression in the sinus mucosa of patients with CRS and NP. The results of immunohistochemistry and RT-qPCR were consistent with each other. Hematoxylin and eosin staining revealed notable lesions in the mucous membranes, goblet cells and cilia of sinus mucosa samples from patients with CRS and NP. The negative correlation between CC10 and TFF1 expression during the progression of CRS and NP suggest that CC10 and TFF1 may serve important roles in its pathogenesis. PMID:29456658

LC-MS guided isolation of ent-kaurane diterpenoids from Nouelia insignis.

PubMed

Sun, Chang-Li; Geng, Chang-An; Chen, Xing-Long; Yang, Tong-Hua; Yin, Xiu-Juan; Huang, Xiao-Yan; Peng, Hua; Chen, Ji-Jun

2016-06-01

The preliminary LC-MS investigation on the stems of Nouelia insignis manifested the existence of diterpenoids. As a result, 15 ent-kaurane diterpenoids, including 7 new glycosides (nouelosides A-G, 1-7), were isolated under the direction of LC-MS analysis. The new compounds were determined by extensive spectroscopic analysis including HRESIMS, 1D and 2D NMR data and chemical methods. Compounds 6 and 15 with the exo-methylene cyclopentanone functional group exhibited obvious nitric oxide production inhibitory activity with IC50 values of 3.84±0.20 and 3.19±0.25μM. Copyright © 2016 Elsevier B.V. All rights reserved.

New application of superconductors: High sensitivity cryogenic light detectors

NASA Astrophysics Data System (ADS)

Cardani, L.; Bellini, F.; Casali, N.; Castellano, M. G.; Colantoni, I.; Coppolecchia, A.; Cosmelli, C.; Cruciani, A.; D'Addabbo, A.; Di Domizio, S.; Martinez, M.; Tomei, C.; Vignati, M.

2017-02-01

In this paper we describe the current status of the CALDER project, which is developing ultra-sensitive light detectors based on superconductors for cryogenic applications. When we apply an AC current to a superconductor, the Cooper pairs oscillate and acquire kinetic inductance, that can be measured by inserting the superconductor in a LC circuit with high merit factor. Interactions in the superconductor can break the Cooper pairs, causing sizable variations in the kinetic inductance and, thus, in the response of the LC circuit. The continuous monitoring of the amplitude and frequency modulation allows to reconstruct the incident energy with excellent sensitivity. This concept is at the basis of Kinetic Inductance Detectors (KIDs) that are characterized by natural aptitude to multiplexed read-out (several sensors can be tuned to different resonant frequencies and coupled to the same line), resolution of few eV, stable behavior over a wide temperature range, and ease in fabrication. We present the results obtained by the CALDER collaboration with 2×2 cm2 substrates sampled by 1 or 4 Aluminum KIDs. We show that the performances of the first prototypes are already competitive with those of other commonly used light detectors, and we discuss the strategies for a further improvement.

The evolutionarily conserved interaction between LC3 and p62 selectively mediates autophagy-dependent degradation of mutant huntingtin.

PubMed

Tung, Ying-Tsen; Hsu, Wen-Ming; Lee, Hsinyu; Huang, Wei-Pang; Liao, Yung-Feng

2010-07-01

Mammalian p62/sequestosome-1 protein binds to both LC3, the mammalian homologue of yeast Atg8, and polyubiquitinated cargo proteins destined to undergo autophagy-mediated degradation. We previously identified a cargo receptor-binding domain in Atg8 that is essential for its interaction with the cargo receptor Atg19 in selective autophagic processes in yeast. We, thus, sought to determine whether this interaction is evolutionally conserved from yeast to mammals. Using an amino acid replacement approach, we demonstrate that cells expressing mutant LC3 (LC3-K30D, LC3-K51A, or LC3-L53A) all exhibit defective lipidation of LC3, a disrupted LC3-p62 interaction, and impaired autophagic degradation of p62, suggesting that the p62-binding site of LC3 is localized within an evolutionarily conserved domain. Importantly, whereas cells expressing these LC3 mutants exhibited similar overall autophagic activity comparable to that of cells expressing wild-type LC3, autophagy-mediated clearance of the aggregation-prone mutant Huntingtin was defective in the mutant-expressing cells. Together, these results suggest that p62 directly binds to the evolutionarily conserved cargo receptor-binding domain of Atg8/LC3 and selectively mediates the clearance of mutant Huntingtin.

A wide-range 22-GHz LC-based CMOS voltage-controlled oscillator

NASA Astrophysics Data System (ADS)

Gharbieh, Karam; Ranneh, Mohammed; Abugharbieh, Khaldoon

2018-06-01

This work presents a novel voltage-controlled oscillator (VCO) design and simulations that combine a varactor bank with a transformer in the LC tank to achieve a high-frequency range. While the varactor bank is responsible for changing the capacitance in the LC tank, the transformer acts as a means to change the value of the inductance, hence allowing tune-ability in the two main components of the VCO. A control mechanism utilises a mixed-mode circuit consisting of comparators and a state machine. It allows efficient tuning of the VCO by controlling the capacitance and transformer in the LC tank. The VCO has a 10.75-22.43 GHz frequency range and the VCO gain, KVCO, is kept at a low value ranging from 98.6 to 175.7 MHz/V. The simulated phase noise is -111 dBc/Hz at 1 MHz offset from the 10.75 GHz oscillation frequency. The circuit is designed and simulated in 28 nm CMOS technology and uses a 1 V supply drawing a typical power of 14.74 mW.

Rational design of botulinum neurotoxin A1 mutants with improved oxidative stability.

PubMed

López de la Paz, Manuela; Scheps, Daniel; Jurk, Marcel; Hofmann, Fred; Frevert, Jürgen

2018-06-01

Botulinum neurotoxins (BoNTs) are the most potent toxic proteins to mankind known but applied in low doses trigger a localized muscle paralysis that is beneficial for the therapy of several neurological disorders and aesthetic treatment. The paralytic effect is generated by the enzymatic activity of the light chain (LC) that cleaves specifically one of the SNARE proteins responsible for neurotransmitter exocytosis. The activity of the LC in a BoNT-containing therapeutic can be compromised by denaturing agents present during manufacturing and/or in the cell. Stabilization of the LC by reducing vulnerability towards denaturants would thus be advantageous for the development of BoNT-based therapeutics. In this work, we focused on increasing the stability of LC of BoNT/A1 (LC/A1) towards oxidative stress. We tackled this task by rational design of mutations at cysteine and methionine LC/A1 sites. Designed mutants showed improved oxidative stability in vitro and equipotency to wildtype toxin in vivo. Our results suggest that suitable modification of the catalytic domain can lead to more stable BoNTs without impairing their therapeutic efficacy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica (Laminariales, Phaeophyta)

NASA Astrophysics Data System (ADS)

Shao, Zhanru; Liu, Fuli; Li, Qiuying; Yao, Jianting; Duan, Delin

2014-03-01

Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments, and consequently may have highly efficient ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity for carbon assimilation. In our study, we cloned the full-length Rubisco gene from S. japonica ( SJ-rbc). It contained an open reading frame for a large subunit gene ( SJ — rbcL) of 1 467 bp, a small subunit gene ( SJ-rbcS) of 420 bp, and a SJ-rbcL/S intergenic spacer of 269 bp. The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa, 5.81 and 15.84 kDa, 4.71, respectively. After induction with 1 mmol/L isopropyl- β-D-thiogalactopyranoside for 5 h and purification by Ni2+ affinity chromatography, electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein. Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night. This observation implied that Rubisco played a role in normal gametophytic growth and development. In juvenile sporophytes, mRNA levels of SJ-rbcL, carbonic anhydrase, Calvin-Benson-Bassham cycle-related enzyme, and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance. Similarly, expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m2·s). Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.

EGY1 plays a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-grown Arabidopsis hypocotyls.

PubMed

Guo, Di; Gao, Xiaorong; Li, Hao; Zhang, Tao; Chen, Gu; Huang, Pingbo; An, Lijia; Li, Ning

2008-03-01

Egy1 was isolated as an ethylene-dependent gravitropism-deficient Arabidopsis mutant. Molecular studies reveal that EGY1 gene encodes a 59-kDa plastid-targeted metalloprotease. It is actively expressed in hypocotyl tissue and targets to endodermal and cortex plastid. Its protein level is up-regulated by both ethylene and light. CAB protein accumulation and chlorophyll level is severely reduced in hypocotyls and endodermal cells, respectively. Sucrose is able to restore the severely reduced starch and lipid contents as well as the deficient endodermal plastid size found in light-grown egy1 hypocotyls yet it fails to rescue the reduced plastid number and chlorophyll level in egy1 endodermal cells. The loss-of-function egy1 mutation results in a smaller size (1.9 +/- 0.3 microm in diameter) and less number (5 +/- 1) of plastids in endodermal cells, which are nearly 50% of the wild-type. EGY1 is specially required for the development of full-size endodermal plastid in seedlings that are grown on sucrose-free media under light. It plays a direct role in controlling the light-induced chlorophyll production, grana formation and plastid replication in endodermal cell. However, it plays an indirect role in regulation of endodermal plastid size. It is likely that the ethylene-dependent gravitropism-deficient phenotype of egy1 hypocotyls may result from the smaller size and less number of endodermal plastids. Gravicurvature assays performed on ethylene-insensitive mutants, etr1-1, etr2-1, ers2-1, ein4-1 and ein2-5, have clearly demonstrated the necessary role for ethylene in vigorous gravitropism of light-grown hypocotyls. The degree of ethylene-dependent gravicurvature is positively correlated with the combined state of endodermal plastid mass and number. Neither ethylene nor EGY1-regulated full-size endodermal plastid is sufficient for promotion of vigorous hypocotyl gravitropism. Presence of 4 full-size plastids per endodermal cell together with ethylene pretreatment of

4-Amino-7-chloroquinolines: probing ligand efficiency provides botulinum neurotoxin serotype A light chain inhibitors with significant antiprotozoal activity

PubMed Central

Opsenica, Igor M.; Tot, Mikloš; Gomba, Laura; Nuss, Jonathan E.; Sciotti, Richard J.; Bavari, Sina; Burnett, James C.; Šolaja, Bogdan A.

2013-01-01

Structurally simplified analogs of dual antimalarial and botulinum neurotoxin serotype A light chain (BoNT/A LC) inhibitor bis-aminoquinoline (1) were prepared. New compounds were designed to improve ligand efficiency while maintaining or exceeding the inhibitory potency of 1. Three of the new compounds are more active than 1 against both indications. Metabolically, the new inhibitors are relatively stable and non-toxic. Twelve, 14, and 15 are more potent BoNT/A LC inhibitors than 1. Additionally, 15 has excellent in vitro antimalarial efficacy, with IC90 values ranging from 4.45-12.11 nM against five Plasmodium falciparum (P.f.) strains: W2, D6, C235, C2A, C2B. The results indicate that the same level of inhibitory efficacy provided by 1 can be retained/exceeded with less structural complexity. Twelve, 14, and 15 provide new platforms for the development of more potent dual BoNT/A LC and P.f. inhibitors adhering to generally accepted chemical properties associated with the druggability of synthetic molecules. PMID:23815186

Accumulation of 19-kDa plasma membrane polypeptide during induction of freezing tolerance in wheat suspension-cultured cells by abscisic acid.

PubMed

Koike, M; Takezawa, D; Arakawa, K; Yoshida, S

1997-06-01

Suspension-cultured cells derived from immature embryos of winter wheat (Triticum aestivum L. cv. Chihoku) were used in experiments designed to obtain clues to the mechanism of the ABA-induced development of freezing tolerance. Cultured cells treated with 50 microM ABA for 5 d at 23 degrees C acquired the maximum level of freezing tolerance (LT50; -21.6 degrees C). The increased freezing tolerance of ABA-treated cells was closely associated with the remarkable accumulation of 19-kDa polypeptides in the plasma membrane. The 19-kDa polypeptide components were isolated by preparative gel electrophoresis and were further separated into one major (AWPM-19) and other minor polypeptide components by Tricine-SDS-PAGE. N-terminal amino acid sequence of AWPM-19 was determined, and a cDNA clone encoding AWPM-19 was isolated by PCR from the library prepared from the ABA-treated cultured cells. The cDNA clone (WPM-1) encoded a 18.9 kDa hydrophobic polypeptide with four putative membrane spanning domains and with a high pI value (10.2). Expression of WPM-1 mRNA was dramatically induced by 50 microM ABA within a few hours. These results suggest that the AWPM-19 might be closely associated with the ABA-induced increase in freezing tolerance in wheat cultured cells.

Quantifying MMA by SLE LC-MS/MS: Unexpected challenges in assay development.

PubMed

Lo, Sheng-Ying; Gordon, Cindy; Sadilkova, Katerina; Jack, Rhona M; Dickerson, Jane A

2016-09-01

Analysis of serum/plasma methylmalonic acid (MMA) is important for the diagnosis and management of methylmalonic acidemia in pediatric populations. This work focuses on developing and validating a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to monitor methylmalonic acidemia using a simple method preparation. MMA and stable isotope labeled d3-MMA was extracted using supported liquid extraction (SLE). Assay imprecision, bias, linearity, recovery and carryover were determined. The relationship between MMA and propionyl acylcarnitine (C3-acylcarnitine) was also evaluated using historical paired results from 51 unique individuals. Baseline separation between MMA and succinic acid was completed in 7min. The assay was linear from 0.1 to 500μM. The intra-day and inter-day imprecision CV ranged from 4.1 to 13.2% (0.3 to 526μM) and 5.0 to 15.7% (0.3 to 233μM), respectively. Recovery ranged from 93 to 125%. The correlation with a national reference laboratory LC-MS/MS assay showed a Deming regression of 1.026 and intercept of -1.335. Carryover was determined to be <0.04%. Patient-specific correlation was observed between MMA and C3-acylcarnitine. This report describes the first LC-MS/MS method using SLE for MMA extraction. In addition, we illustrate the challenges encountered during this method development that should be assessed and resolved by any laboratory implementing a SLE LC-MS/MS assay designed to quantify analytes across several orders of magnitude. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Data reduction of isotope-resolved LC-MS spectra.

PubMed

Du, Peicheng; Sudha, Rajagopalan; Prystowsky, Michael B; Angeletti, Ruth Hogue

2007-06-01

Data reduction of liquid chromatography-mass spectrometry (LC-MS) spectra can be a challenge due to the inherent complexity of biological samples, noise and non-flat baseline. We present a new algorithm, LCMS-2D, for reliable data reduction of LC-MS proteomics data. LCMS-2D can reliably reduce LC-MS spectra with multiple scans to a list of elution peaks, and subsequently to a list of peptide masses. It is capable of noise removal, and deconvoluting peaks that overlap in m/z, in retention time, or both, by using a novel iterative peak-picking step, a 'rescue' step, and a modified variable selection method. LCMS-2D performs well with three sets of annotated LC-MS spectra, yielding results that are better than those from PepList, msInspect and the vendor software BioAnalyst. The software LCMS-2D is available under the GNU general public license from http://www.bioc.aecom.yu.edu/labs/angellab/as a standalone C program running on LINUX.

MALDI-TOF mass spectrometry analysis of small molecular weight compounds (under 10 KDa) as biomarkers of rat hearts undergoing arecoline challenge.

PubMed

Chen, Tung-Sheng; Chang, Mu-Hsin; Kuo, Wei-Wen; Lin, Yueh-Min; Yeh, Yu-Lan; Day, Cecilia Hsuan; Lin, Chien-Chung; Tsai, Fuu-Jen; Tsai, Chang-Hai; Huang, Chih-Yang

2013-04-01

Statistical and clinical reports indicate that betel nut chewing is strongly associated with progression of oral cancer because some ingredients in betel nuts are potential cancer promoters, especially arecoline. Early diagnosis for cancer biomarkers is the best strategy for prevention of cancer progression. Several methods are suggested for investigating cancer biomarkers. Among these methods, gel-based proteomics approach is the most powerful and recommended tool for investigating biomarkers due to its high-throughput. However, this proteomics approach is not suitable for screening biomarkers with molecular weight under 10 KDa because of the characteristics of gel electrophoresis. This study investigated biomarkers with molecular weight under 10 KDa in rats with arecoline challenge. The centrifuging vials with membrane (10 KDa molecular weight cut-off) played a crucial role in this study. After centrifuging, the filtrate (containing compounds with molecular weight under 10 KDa) was collected and spotted on a sample plate for MALDI-TOF mass spectrometry analysis. Compared to control, three extra peaks (m/z values were 1553.1611, 1668.2097 and 1740.1832, respectively) were found in sera and two extra peaks were found in heart tissue samples (408.9719 and 524.9961, respectively). These small compounds should play important roles and may be potential biomarker candidates in rats with arecoline. This study successfully reports a mass-based method for investigating biomarker candidates with small molecular weight in different types of sample (including serum and tissue). In addition, this reported method is more time-efficient (1 working day) than gel-based proteomics approach (5~7 working days).

Current anti-myeloma therapies in renal manifestations of monoclonal light chain-associated Fanconi syndrome: a retrospective series of 49 patients.

PubMed

Vignon, M; Javaugue, V; Alexander, M P; El-Karoui, K; Karras, A; Roos-Weil, D; Royer, B; Asli, B; Knebelmann, B; Touchard, G; Jaccard, A; Arnulf, B; Bridoux, F; Leung, N; Fermand, J P

2017-01-01

We retrospectively reviewed 49 patients with light chain (LC) Fanconi syndrome (FS). Patients presented with chronic kidney disease (median estimated glomerular filtration rate (eGFR) of 33 ml/min/1.73 m 2 ) and tubular proteinuria. All patients tested had elevated fractional excretion of phosphate, uric acid, generalized aminoaciduria and/or normoglycemic glycosuria. Thirty-eight patients had monoclonal gammopathy of renal significance and eleven patients had an overt hematological malignancy. The monoclonal LC isotype was kappa in 46/49 cases. Kidney biopsy in 39 patients showed various proximal tubular lesions and characteristic LC intracytoplasmic crystalline inclusions in 24 patients. Forty-two patients received chemotherapy. Patients with plasma cell proliferation (n=38) received bortezomib-based regimens (n=11), immunomodulatory agents (n=7) or alkylating agents (n=6). High-dose melphalan (HDM) followed by autologous stem cell transplantation was performed in 14 patients. Hematological response was obtained in 90% of evaluable patients, assessed on serum free light chains (FLC). GFR remained stable as long as hematological response was maintained and declined when serum FLC level rebounded. Improvement in proximal tubule function occurred in 13 patients. In patients with LC-associated FS, chemotherapy using HDM and/or new generation anti-myeloma agents can stabilize renal function and improve proximal tubule function. Serum FLC should be used to assess the hematological response, related to renal outcome.

Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, J.; Varner, J.E.

1985-07-01

Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 asmore » a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding.« less

The autophagy marker LC3 strongly predicts immediate mortality after surgical resection for hepatocellular carcinoma.

PubMed

Lin, Chih-Wen; Lin, Chih-Che; Lee, Po-Huang; Lo, Gin-Ho; Hsieh, Pei-Min; Koh, Kah Wee; Lee, Chih-Yuan; Chen, Yao-Li; Dai, Chia-Yen; Huang, Jee-Fu; Chuang, Wang-Long; Chen, Yaw-Sen; Yu, Ming-Lung

2017-11-03

The remnant liver's ability to regenerate may affect post-hepatectomy immediate mortality. The promotion of autophagy post-hepatectomy could enhance liver regeneration and reduce mortality. This study aimed to identify predictive factors of immediate mortality after surgical resection for hepatocellular carcinoma (HCC). A total of 535 consecutive HCC patients who had undergone their first surgical resection in Taiwan were enrolled between 2010 and 2014. Clinicopathological data and immediate mortality, defined as all cause-mortality within three months after surgery, were analyzed. The expression of autophagy proteins (LC3, Beclin-1, and p62) in adjacent non-tumor tissues was scored by immunohistochemical staining. Approximately 5% of patients had immediate mortality after surgery. The absence of LC3, hypoalbuminemia (<3.5 g/dl), high alanine aminotransferase, and major liver surgery were significantly associated with immediate mortality in univariate analyses. Multivariate logistic regression demonstrated that absence of LC3 (hazard ratio/95% confidence interval: 40.8/5.14-325) and hypoalbuminemia (2.88/1.11-7.52) were significantly associated with immediate mortality. The 3-month cumulative incidence of mortality was 12.1%, 13.0%, 21.4% and 0.4%, respectively, among patients with absence of LC3 expression, hypoalbuminemia, both, or neither of the two. In conclusion, the absence of LC3 expression in adjacent non-tumor tissues and hypoalbuminemia were strongly predictive of immediate mortality after resection for HCC.

Host and bacterial factors that regulate LC3 recruitment to Listeria monocytogenes during the early stages of macrophage infection.

PubMed

Lam, Grace Y; Cemma, Marija; Muise, Aleixo M; Higgins, Darren E; Brumell, John H

2013-07-01

Listeria monocytogenes is a bacterial pathogen that can escape the phagosome and replicate in the cytosol of host cells during infection. We previously observed that a population (up to 35%) of L. monocytogenes strain 10403S colocalize with the macroautophagy marker LC3 at 1 h postinfection. This is thought to give rise to spacious Listeria-containing phagosomes (SLAPs), a membrane-bound compartment harboring slow-growing bacteria that is associated with persistent infection. Here, we examined the host and bacterial factors that mediate LC3 recruitment to bacteria at 1 h postinfection. At this early time point, LC3(+) bacteria were present within single-membrane phagosomes that are LAMP1(+). Protein ubiquitination is known to play a role in targeting cytosolic L. monocytogenes to macroautophagy. However, we found that neither protein ubiquitination nor the ubiquitin-binding adaptor SQSTM1/p62 are associated with LC3(+) bacteria at 1 h postinfection. Reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase was also required for LC3 recruitment to bacteria at 1 h postinfection and for subsequent SLAP formation. Diacylglycerol is an upstream activator of the CYBB/NOX2 NADPH oxidase, and its production by both bacterial and host phospholipases was required for LC3 recruitment to bacteria. Our data suggest that the LC3-associated phagocytosis (LAP) pathway, which is distinct from macroautophagy, targets L. monocytogenes during the early stage of infection within host macrophages and allows establishment of an intracellular niche (SLAPs) associated with persistent infection.

Dissecting the involvement of LC3B and GATE-16 in p62 recruitment into autophagosomes.

PubMed

Shvets, Elena; Abada, Adi; Weidberg, Hilla; Elazar, Zvulun

2011-07-01

Autophagy is a major intracellular trafficking pathway that delivers proteins and organelles from the cytoplasm into lysosomes for consequential degradation and recycling. Mammalian Atg8s are key autophagic factors that undergo a unique ubiquitin-like conjugation to the lipid phase of the autophagosomal membrane. In addition to their activity in autophagosome formation, several Atg8s directly bind p62/SQSTM1. Here we show that LC3 and GATE-16 differ in their mode of p62 binding. While the soluble form of both LC3 and GATE-16 bind p62, only the lipidated form of LC3 is directly involved in p62 recruitment into autophagosomes. Moreover, by utilizing chimeras of LC3 and GATE-16 where their N-terminus was swapped, we determined the regions responsible for this differential binding. Accordingly, we found that the chimera of GATE-16 containing the LC3 N-terminal region acts similarly to wild-type LC3 in recruiting p62 into autophagosomes. We therefore propose that LC3 is responsible for the final stages of p62 incorporation into autophagosomes, a process selectively mediated by its N-terminus.

Development of the foremost light-curable calcium-silicate MTA cement as root-end in oral surgery. Chemical-physical properties, bioactivity and biological behavior.

PubMed

Gandolfi, Maria Giovanna; Taddei, Paola; Siboni, Francesco; Modena, Enrico; Ciapetti, Gabriela; Prati, Carlo

2011-07-01

An innovative light-curable calcium-silicate cement containing a HEMA-TEGDMA-based resin (lc-MTA) was designed to obtain a bioactive fast setting root-end filling and root repair material. lc-MTA was tested for setting time, solubility, water absorption, calcium release, alkalinizing activity (pH of soaking water), bioactivity (apatite-forming ability) and cell growth-proliferation. The apatite-forming ability was investigated by micro-Raman, ATR-FTIR and ESEM/EDX after immersion at 37°C for 1-28 days in DPBS or DMEM+FBS. The marginal adaptation of cement in root-end cavities of extracted teeth was assessed by ESEM/EDX, and the viability of Saos-2 cell on cements was evaluated. lc-MTA demonstrated a rapid setting time (2min), low solubility, high calcium release (150-200ppm) and alkalinizing power (pH 10-12). lc-MTA proved the formation of bone-like apatite spherulites just after 1 day. Apatite precipitates completely filled the interface porosities and created a perfect marginal adaptation. lc-MTA allowed Saos-2 cell viability and growth and no compromising toxicity was exerted. HEMA-TEGDMA creates a polymeric network able to stabilize the outer surface of the cement and a hydrophilic matrix permeable enough to allow water absorption. SiO(-)/Si-OH groups from the mineral particles induce heterogeneous nucleation of apatite by sorption of calcium and phosphate ions. Oxygen-containing groups from poly-HEMA-TEGDMA provide additional apatite nucleating sites through the formation of calcium chelates. The strong novelty was that the combination of a hydraulic calcium-silicate powder and a poly-HEMA-TEGDMA hydrophilic resin creates the conditions (calcium release and functional groups able to chelate Ca ions) for a bioactive fast setting light-curable material for clinical applications in dental and maxillofacial surgery. The first and unique/exclusive light-curable calcium-silicate MTA cement for endodontics and root-end application was created, with a potential

Analytical Stability-Indicating Methods for Alogliptin in Tablets by LC-CAD and LC-UV.

PubMed

Bertol, Charise Dallazem; Friedrich, Maria Tereza; Carlos, Graciela; Froehlich, Pedro Eduardo

2017-03-01

Stability-indicating LC methods using a UV detector and a charged aerosol detector (CAD) simultaneously were validated for the assessment of alogliptin (ALG) in tablets. The analysis was performed on a C8 column (250 × 4.6 mm, 5 μm) at a flow of 0.8 mL/min, using acetonitrile-10 mM ammonium acetate buffer (pH 3.5; 90 + 10, v/v) as mobile phase and UV detection at 275 nm. Validation followed the International Conference on Harmonization guidelines. The method was linear over the range of 25-200 μg/mL. Normality of the residuals showed a normal distribution, no autocorrelation, and homoscedasticity. LODs were 6.25 and 2.65 µg/mL and LOQs were 20.85 and 8.84 µg/mL for the CAD and the UV detector, respectively. The methods were precise and accurate. Excipients and degradation products did not interfere in the methods in studies of specificity. None of the factors studied in the analysis of robustness had a significant effect on the quantification of the ALG by the Pareto chart. The results of the assay obtained with LC-CAD and LC-UV were similar. The methods could be considered interchangeable and stability-indicating, and can be applied as an appropriate QC tool for analysis of ALG in tablets.

The glyoxysomal and plastid molecular chaperones (70-kDa heat shock protein) of watermelon cotyledons are encoded by a single gene

PubMed Central

Wimmer, Bernhard; Lottspeich, Friedrich; van der Klei, Ida; Veenhuis, Marten; Gietl, Christine

1997-01-01

The monoclonal a-70-kDa heat shock protein (hsp70) antibody recognizes in crude extracts from watermelon (Citrullus vulgaris) cotyledons two hsps with molecular masses of 70 and 72 kDa. Immunocytochemistry on watermelon cotyledon tissue and on isolated glyoxysomes identified hsp70s in the matrix of glyoxysomes and plastids. Affinity purification and partial amino acid determination revealed the 70-kDa protein to share high sequence identity with cytosolic hsp70s from a number of plant species, while the 72 kDa protein was very similar to plastid hsp70s from pea and cucumber. A full-length cDNA clone encoding the 72-kDa hsp70 was isolated and identified two start methionines in frame within the N-terminal presequence leading either to an N-terminal extension of 67 amino acids or to a shorter one of 47 amino acids. The longer presequence was necessary and sufficient to target a reporter protein into watermelon proplastids in vitro. The shorter extension starting from the second methionine within the long version harbored a consensus peroxisomal targeting signal (RT-X5-KL) that directed in vivo a reporter protein into peroxisomes of the yeast Hansenula polymorpha. Peroxisomal targeting was however prevented, when the 67-residue presequence was fused to the reporter protein, indicating that the peroxisomal targeting signal 2 information is hidden in this context. We propose that the 72-kDa hsp70 is encoded by a single gene, but targeted alternatively into two organelles by the modulated use of its presequence. PMID:9391076

[Stimulation of cell cultures recovery after cryopreservation by the cattle cord blood FRACTION (below 5 kDa) or Actovegin].

PubMed

Gulevskiĭ, A K; Trifonova, A V; Lavrik, A A

2013-01-01

The capacities of the cattle cord blood low-molecular fraction (below 5 kDa) and Actovegin (the vealer blood fraction (below 5 kDa)) for recovering functions of cell cultures after cryopreservation compared. Their influence proliferation of the flozen-thawed cell cultures, certain stages of their growth, cell attachment, rate of cell spreading, and mitotic regiment has been studied. Both the cord blood low-molecular fraction and Actovegin were shown to stimulate growth of the cell cultures after cryopreservation more efficiently at the concentration of 224 μg/ml. However, despite the stimulating effect discovered, their application did not bring proliferative indices on the 1st passage after cryopreservation to the values of the native culture. The effects of the cord blood low-molecular fraction and Actovegin on the human fibroblast culture were identical by the following parameters: cell attachment, rates of cell spreading and proliferation. In culture BHK-21 clone 13/04 the efficiency of Actovegin was low, while the cord blood low-molecular fraction has a conspicuous stimulating effect on its adhesion and proliferation. The investigations carried out can serve as a basis for the development of regenerative media containing the cattle cord blood low-molecular fraction (below 5 kDa) or Actovegin as active components at the concentration of 224 μg/ml with the purpose of fast recovery of culture prolifetative properties after cryopreservation.

Hepatitis C virus core protein activates autophagy through EIF2AK3 and ATF6 UPR pathway-mediated MAP1LC3B and ATG12 expression

PubMed Central

Wang, Ji; Kang, Rongyan; Huang, He; Xi, Xueyan; Wang, Bei; Wang, Jianwei; Zhao, Zhendong

2014-01-01

HCV infection induces autophagy, but how this occurs is unclear. Here, we report the induction of autophagy by the structural HCV core protein and subsequent endoplasmic reticular (ER) stress in Huh7 hepatoma cells. During ER stress, both the EIF2AK3 and ATF6 pathways of the unfolded protein response (UPR) were activated by HCV core protein. Then, these pathways upregulated transcription factors ATF4 and DDIT3. The ERN1-XBP1 pathway was not activated. Through ATF4 in the EIF2AK3 pathway, the autophagy gene ATG12 was upregulated. DDIT3 upregulated the transcription of autophagy gene MAP1LC3B (LC3B) by directly binding to the –253 to –99 base region of the LC3B promoter, contributing to the development of autophagy. Collectively, these data suggest not only a novel role for the HCV core protein in autophagy but also offer new insight into detailed molecular mechanisms with respect to HCV-induced autophagy, specifically how downstream UPR molecules regulate key autophagic gene expression. PMID:24589849

Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori.

PubMed

Jeong, Kyoung Yong; Son, Mina; Lee, June Yong; Park, Kyung Hee; Lee, Jae-Hyun; Park, Jung-Won

2016-01-01

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.

Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori

PubMed Central

Son, Mina; Lee, June Yong

2016-01-01

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm. PMID:26770033

Monoclonal antibodies against 27.8 kDa protein receptor efficiently block lymphocystis disease virus infection in flounder Paralichthys olivaceus gill cells.

PubMed

Sheng, Xiu-Zhen; Wang, Mu; Xing, Jing; Zhan, Wen-Bin

2012-08-13

In previous research using co-immunoprecipitation, a 27.8 kDa protein in flounder Paralichthys olivaceus gill (FG) cells was found to bind lymphocystis disease virus (LCDV). In this paper, 13 hybridomas secreting monoclonal antibodies (MAbs) against the 27.8 kDa protein were obtained, and 2 MAbs designated as 2G11 and 3D9 were cloned by limiting dilution. Analyzed by indirect enzyme-linked immunosorbent assay (ELISA) and western blotting, the MAbs specifically reacted with the 27.8 kDa protein of FG cells. Confocal fluorescence microscopy and immunogold electron microscopy (IEM) provided evidence that the epitopes recognized by these MAbs were located primarily on the cell membrane and occasionally in the cytoplasm near the cell membrane of FG cells. The MAbs could block LCDV binding after MAbs were pre-incubated with isolated membrane proteins of FG cells in a blocking ELISA, and MAbs also could inhibit LCDV infection of FG cells in culture. Moreover, several target tissues of LCDV in flounder, including gill, stomach, intestine and liver, displayed the presence of the LCDV receptor-27.8 kDa. These results strongly supported the possibility that the 27.8 kDa protein is the putative receptor specific for LCDV infection of FG cells in flounder.

Quantum dots-based immunofluorescent imaging of stromal fibroblasts Caveolin-1 and light chain 3B expression and identification of their clinical significance in human gastric cancer.

PubMed

He, Yuyu; Zhao, Xianda; Gao, Jun; Fan, Lifang; Yang, Guifang; Cho, William Chi-Shing; Chen, Honglei

2012-10-24

Caveolin-1 (Cav-1) expression deficiency and autophagy in tumor stromal fibroblasts (hereafter fibroblasts) are involved in tumor proliferation and progression, particularly in breast and prostate cancer. The aim of this study was to detect the expression of fibroblastic Cav-1 and LC3B, markers of autophagy, in gastric cancer (GC) and to analyze their clinical significances. Furthermore, because Epstein-Barr virus (EBV)-associated GC (EBVaGC) is a unique subtype of GC; we compared the differential expression of fibroblastic Cav-1 and LC3B in EBVaGC and non-EBVaGC. Quantum dots (QDs)-based immunofluorescence histochemistry was used to examine the expression of fibroblastic Cav-1 and LC3B in 118 cases of GC with adequate stroma. QDs-based double immunofluorescence labeling was performed to detect the coexpression of Cav-1 and LC3B proteins. EBV-encoded small RNA was detected by QDs-based fluorescence in situ hybridization to identify EBVaGC. Multivariate analysis indicated that low fibroblastic Cav-1 level was an independent prognosticator (p = 0.029) that predicted poorer survival of GC patients. Positive fibroblastic LC3B was correlated with lower invasion (p = 0.032) and was positively associated with Cav-1 expression (r = 0.432, p < 0.001). EBV infection did not affect fibroblastic Cav-1 and LC3B expression. In conclusion, positive fibroblastic LC3B correlates with lower invasion, and low expression of fibroblastic Cav-1 is a novel predictor of poor GC prognosis.

A novel PGC-1α isoform in brain localizes to mitochondria and associates with PINK1 and VDAC

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Joungil, E-mail: jochoi@som.umaryland.edu; Veterans Affairs Medical Center, Baltimore, MD 21201; Batchu, Vera Venkatanaresh Kumar

2013-06-14

Highlights: •Novel 35 kDa PGC-1α localizes to mitochondrial inner membrane and matrix in brain. •Mitochondrial localization of 35 kDa PGC-1α depends on VDAC protein. •Mitochondrial localization of 35 kDa PGC-1α depends on membrane potential. •The 35 kDa PGC-1α associates and colocalizes with PINK in brain mitochondria. -- Abstract: Peroxisome proliferator-activated receptor-gamma co-activator 1α (PGC-1α) and PTEN-induced putative kinase 1 (PINK1) are powerful regulators of mitochondrial function. Here, we report that a previously unrecognized, novel 35 kDa PGC-1α isoform localizes to the mitochondrial inner membrane and matrix in brain as determined by protease protection and carbonate extraction assays, as well asmore » by immunoelectron microscopy. Immunoelectron microscopy and import experiments in vitro revealed that 35 kDa PGC-1α colocalizes and interacts with the voltage-dependent anion channel (VDAC), and that its import depends on VDAC. Valinomycin treatment which depolarizes the membrane potential, abolished mitochondrial localization of the 35 kDa PGC-1α. Using blue native-PAGE, co-immunoprecipitation, and immunoelectron microscopy analyses, we found that the 35 kDa PGC-1α binds and colocalizes with PINK1 in brain mitochondria. This is the first report regarding mitochondrial localization of a novel 35 kDa PGC-1α isoform and its association with PINK1, suggesting possible regulatory roles for mitochondrial function in the brain.« less

Visible light high-resolution imaging system for large aperture telescope by liquid crystal adaptive optics with phase diversity technique.

PubMed

Xu, Zihao; Yang, Chengliang; Zhang, Peiguang; Zhang, Xingyun; Cao, Zhaoliang; Mu, Quanquan; Sun, Qiang; Xuan, Li

2017-08-30

There are more than eight large aperture telescopes (larger than eight meters) equipped with adaptive optics system in the world until now. Due to the limitations such as the difficulties of increasing actuator number of deformable mirror, most of them work in the infrared waveband. A novel two-step high-resolution optical imaging approach is proposed by applying phase diversity (PD) technique to the open-loop liquid crystal adaptive optics system (LC AOS) for visible light high-resolution adaptive imaging. Considering the traditional PD is not suitable for LC AOS, the novel PD strategy is proposed which can reduce the wavefront estimating error caused by non-modulated light generated by liquid crystal spatial light modulator (LC SLM) and make the residual distortions after open-loop correction to be smaller. Moreover, the LC SLM can introduce any aberration which realizes the free selection of phase diversity. The estimating errors are greatly reduced in both simulations and experiments. The resolution of the reconstructed image is greatly improved on both subjective visual effect and the highest discernible space resolution. Such technique can be widely used in large aperture telescopes for astronomical observations such as terrestrial planets, quasars and also can be used in other applications related to wavefront correction.

Purification, Characterization and Analysis of the Allergenic Properties of Myosin Light Chain in Procambarus clarkia.

USDA-ARS?s Scientific Manuscript database

Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in close species. In this study, MLC with the molecular mass of 18kDa was purified from crayfish (Procambarus clarkii) muscle fibrils. Its physicochemical characterization showed that the...

Ultratight crystal packing of a 10 kDa protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trillo-Muyo, Sergio; Jasilionis, Andrius; Domagalski, Marcin J.

2013-03-01

The crystal structure of the C-terminal domain of a putative U32 peptidase from G. thermoleovorans is reported; it is one of the most tightly packed protein structures reported to date. While small organic molecules generally crystallize forming tightly packed lattices with little solvent content, proteins form air-sensitive high-solvent-content crystals. Here, the crystallization and full structure analysis of a novel recombinant 10 kDa protein corresponding to the C-terminal domain of a putative U32 peptidase are reported. The orthorhombic crystal contained only 24.5% solvent and is therefore among the most tightly packed protein lattices ever reported.

An LC-IMS-MS Platform Providing Increased Dynamic Range for High-Throughput Proteomic Studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baker, Erin Shammel; Livesay, Eric A.; Orton, Daniel J.

2010-02-05

A high-throughput approach and platform using 15 minute reversed-phase capillary liquid chromatography (RPLC) separations in conjunction with ion mobility spectrometry-mass spectrometry (IMS-MS) measurements was evaluated for the rapid analysis of complex proteomics samples. To test the separation quality of the short LC gradient, a sample was prepared by spiking twenty reference peptides at varying concentrations from 1 ng/mL to 10 µg/mL into a tryptic digest of mouse blood plasma and analyzed with both a LC-Linear Ion Trap Fourier Transform (FT) MS and LC-IMS-TOF MS. The LC-FT MS detected thirteen out of the twenty spiked peptides that had concentrations ≥100 ng/mL.more » In contrast, the drift time selected mass spectra from the LC-IMS-TOF MS analyses yielded identifications for nineteen of the twenty peptides with all spiking level present. The greater dynamic range of the LC-IMS-TOF MS system could be attributed to two factors. First, the LC-IMS-TOF MS system enabled drift time separation of the low concentration spiked peptides from the high concentration mouse peptide matrix components, reducing signal interference and background, and allowing species to be resolved that would otherwise be obscured by other components. Second, the automatic gain control (AGC) in the linear ion trap of the hybrid FT MS instrument limits the number of ions that are accumulated to reduce space charge effects, but in turn limits the achievable dynamic range compared to the TOF detector.« less

Contaminant screening of wastewater with HPLC-IM-qTOF-MS and LC+LC-IM-qTOF-MS using a CCS database.

PubMed

Stephan, Susanne; Hippler, Joerg; Köhler, Timo; Deeb, Ahmad A; Schmidt, Torsten C; Schmitz, Oliver J

2016-09-01

Non-target analysis has become an important tool in the field of water analysis since a broad variety of pollutants from different sources are released to the water cycle. For identification of compounds in such complex samples, liquid chromatography coupled to high resolution mass spectrometry are often used. The introduction of ion mobility spectrometry provides an additional separation dimension and allows determining collision cross sections (CCS) of the analytes as a further physicochemical constant supporting the identification. A CCS database with more than 500 standard substances including drug-like compounds and pesticides was used for CCS data base search in this work. A non-target analysis of a wastewater sample was initially performed with high performance liquid chromatography (HPLC) coupled to an ion mobility-quadrupole-time of flight mass spectrometer (IM-qTOF-MS). A database search including exact mass (±5 ppm) and CCS (±1 %) delivered 22 different compounds. Furthermore, the same sample was analyzed with a two-dimensional LC method, called LC+LC, developed in our group for the coupling to IM-qTOF-MS. This four dimensional separation platform revealed 53 different compounds, identified over exact mass and CCS, in the examined wastewater sample. It is demonstrated that the CCS database can also help to distinguish between isobaric structures exemplified for cyclophosphamide and ifosfamide. Graphical Abstract Scheme of sample analysis and database screening.

The patient education - Learning and Coping Strategies - improves adherence in cardiac rehabilitation (LC-REHAB): A randomised controlled trial.

PubMed

Lynggaard, Vibeke; Nielsen, Claus Vinther; Zwisler, Ann-Dorthe; Taylor, Rod S; May, Ole

2017-06-01

Despite proven benefits of cardiac rehabilitation (CR), adherence to CR remains suboptimal. This trial aimed to assess the impact of the patient education 'Learning and Coping Strategies' (LC) on patient adherence to an eight-week CR program. 825 patients with ischaemic heart disease or heart failure were open label randomised to either the LC arm (LC plus CR) or the control arm (CR alone) across three hospital units in Denmark. Both arms received same amount of training and education hours. LC consisted of individual clarifying interviews, participation of experienced patients as co-educators, situational, reflective and inductive teaching. The control arm received structured deductive teaching. The primary outcomes were patient adherence to at least 75% of the exercise training or education sessions. We tested for subgroup effects on the primary outcomes using interaction terms. The primary outcomes were compared across arms using logistic regression. More patients in the LC arm adhered to at least 75% of the exercise training sessions than control (80% versus 73%, adjusted odds ratio (OR):1.48; 95% CI:1.07 to 2.05, P=0.018) and 75% of education sessions (79% versus 70%, adjusted OR:1.61, 1.17 to 2.22, P=0.003). Some evidence of larger effects of LC on adherence was seen for patients with heart failure, low education and household income. Addition of LC strategies improved adherence in rehabilitation both in terms of exercise training and education. Patients with heart failure, low levels of education and household income appear to benefit most from this adherence promoting intervention. www.clinicaltrials.gov identifier NCT01668394. Copyright © 2017 Elsevier B.V. All rights reserved.

4-hydroxyphenylacetic acid derivatives of inositol from dandelion (Taraxacum officinale) root characterised using LC-SPE-NMR and LC-MS techniques.

PubMed

Kenny, O; Smyth, T J; Hewage, C M; Brunton, N P; McLoughlin, P

2014-02-01

The combination of hyphenated techniques, LC-SPE-NMR and LC-MS, to isolate and identify minor isomeric compounds from an ethyl acetate fraction of Taraxacum officinale root was employed in this study. Two distinct fractions of 4-hydroxyphenylacetic acid derivatives of inositol were isolated and characterised by spectroscopic methods. The (1)H NMR spectra and MS data revealed two groups of compounds, one of which were derivatives of the di-4-hydroxyphenylacetic acid derivative of the inositol compound tetrahydroxy-5-[2-(4-hydroxyphenyl)acetyl] oxycyclohexyl-2-(4-hydroxyphenyl) acetate, while the other group consisted of similar tri-substituted inositol derivatives. For both fractions the derivatives of inositols vary in the number of 4-hydroxyphenylacetic acid groups present and their position and geometry on the inositol ring. In total, three di-substituted and three tri-substituted 4-hydroxyphenylacetic acid inositol derivates were identified for the first time along with a further two previously reported di-substituted inositol derivatives. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF

PubMed Central

Jiang, Hui; Sidhu, Rohini; Fujiwara, Hideji; De Meulder, Marc; de Vries, Ronald; Gong, Yong; Kao, Mark; Porter, Forbes D.; Yanjanin, Nicole M.; Carillo-Carasco, Nuria; Xu, Xin; Ottinger, Elizabeth; Woolery, Myra; Ory, Daniel S.; Jiang, Xuntian

2014-01-01

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and “dilute and shoot” procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients. PMID:24868096

46 CFR 111.75-1 - Lighting feeders.

Code of Federal Regulations, 2010 CFR

2010-10-01

... COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 Lighting feeders. (a) Passenger vessels. On a... lighting, feeders, and branch circuits are in subpart 112.43 of this chapter. [CGD 74-125A, 47 FR 15236...

Human Atg8-cardiolipin interactions in mitophagy: Specific properties of LC3B, GABARAPL2 and GABARAP.

PubMed

Antón, Zuriñe; Landajuela, Ane; Hervás, Javier H; Montes, L Ruth; Hernández-Tiedra, Sonia; Velasco, Guillermo; Goñi, Felix M; Alonso, Alicia

2016-12-01

The phospholipid cardiolipin (CL) has been proposed to play a role in selective mitochondrial autophagy, or mitophagy. CL externalization to the outer mitochondrial membrane would act as a signal for the human Atg8 ortholog subfamily, MAP1LC3 (LC3). The latter would mediate both mitochondrial recognition and autophagosome formation, ultimately leading to removal of damaged mitochondria. We have applied quantitative biophysical techniques to the study of CL interaction with various Atg8 human orthologs, namely LC3B, GABARAPL2 and GABARAP. We have found that LC3B interacts preferentially with CL over other di-anionic lipids, that CL-LC3B binding occurs with positive cooperativity, and that the CL-LC3B interaction relies only partially on electrostatic forces. CL-induced increased membrane fluidity appears also as an important factor helping LC3B to bind CL. The LC3B C terminus remains exposed to the hydrophilic environment after protein binding to CL-enriched membranes. In intact U87MG human glioblastoma cells rotenone-induced autophagy leads to LC3B translocation to mitochondria and subsequent delivery of mitochondria to lysosomes. We have also observed that GABARAP, but not GABARAPL2, interacts with CL in vitro. However neither GABARAP nor GABARAPL2 were translocated to mitochondria in rotenone-treated U87MG cells. Thus the various human Atg8 orthologs might play specific roles in different autophagic processes.

15. INTERIOR OF SOUTH BEDROOM SHOWING 6LIGHT OVER 1LIGHT, DOUBLEHUNG, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

15. INTERIOR OF SOUTH BEDROOM SHOWING 6-LIGHT OVER 1-LIGHT, DOUBLE-HUNG, WOOD-FRAME WINDOWS ON EAST AND SOUTH WALL. VIEW TO SOUTHWEST. - Bishop Creek Hydroelectric System, Plant 4, Worker Cottage, Bishop Creek, Bishop, Inyo County, CA

Profile-Based LC-MS Data Alignment—A Bayesian Approach

PubMed Central

Tsai, Tsung-Heng; Tadesse, Mahlet G.; Wang, Yue; Ressom, Habtom W.

2014-01-01

A Bayesian alignment model (BAM) is proposed for alignment of liquid chromatography-mass spectrometry (LC-MS) data. BAM belongs to the category of profile-based approaches, which are composed of two major components: a prototype function and a set of mapping functions. Appropriate estimation of these functions is crucial for good alignment results. BAM uses Markov chain Monte Carlo (MCMC) methods to draw inference on the model parameters and improves on existing MCMC-based alignment methods through 1) the implementation of an efficient MCMC sampler and 2) an adaptive selection of knots. A block Metropolis-Hastings algorithm that mitigates the problem of the MCMC sampler getting stuck at local modes of the posterior distribution is used for the update of the mapping function coefficients. In addition, a stochastic search variable selection (SSVS) methodology is used to determine the number and positions of knots. We applied BAM to a simulated data set, an LC-MS proteomic data set, and two LC-MS metabolomic data sets, and compared its performance with the Bayesian hierarchical curve registration (BHCR) model, the dynamic time-warping (DTW) model, and the continuous profile model (CPM). The advantage of applying appropriate profile-based retention time correction prior to performing a feature-based approach is also demonstrated through the metabolomic data sets. PMID:23929872

ICPD-a new peak detection algorithm for LC/MS.

PubMed

Zhang, Jianqiu; Haskins, William

2010-12-01

The identification and quantification of proteins using label-free Liquid Chromatography/Mass Spectrometry (LC/MS) play crucial roles in biological and biomedical research. Increasing evidence has shown that biomarkers are often low abundance proteins. However, LC/MS systems are subject to considerable noise and sample variability, whose statistical characteristics are still elusive, making computational identification of low abundance proteins extremely challenging. As a result, the inability of identifying low abundance proteins in a proteomic study is the main bottleneck in protein biomarker discovery. In this paper, we propose a new peak detection method called Information Combining Peak Detection (ICPD ) for high resolution LC/MS. In LC/MS, peptides elute during a certain time period and as a result, peptide isotope patterns are registered in multiple MS scans. The key feature of the new algorithm is that the observed isotope patterns registered in multiple scans are combined together for estimating the likelihood of the peptide existence. An isotope pattern matching score based on the likelihood probability is provided and utilized for peak detection. The performance of the new algorithm is evaluated based on protein standards with 48 known proteins. The evaluation shows better peak detection accuracy for low abundance proteins than other LC/MS peak detection methods.

ICPD-A New Peak Detection Algorithm for LC/MS

PubMed Central

2010-01-01

Background The identification and quantification of proteins using label-free Liquid Chromatography/Mass Spectrometry (LC/MS) play crucial roles in biological and biomedical research. Increasing evidence has shown that biomarkers are often low abundance proteins. However, LC/MS systems are subject to considerable noise and sample variability, whose statistical characteristics are still elusive, making computational identification of low abundance proteins extremely challenging. As a result, the inability of identifying low abundance proteins in a proteomic study is the main bottleneck in protein biomarker discovery. Results In this paper, we propose a new peak detection method called Information Combining Peak Detection (ICPD ) for high resolution LC/MS. In LC/MS, peptides elute during a certain time period and as a result, peptide isotope patterns are registered in multiple MS scans. The key feature of the new algorithm is that the observed isotope patterns registered in multiple scans are combined together for estimating the likelihood of the peptide existence. An isotope pattern matching score based on the likelihood probability is provided and utilized for peak detection. Conclusions The performance of the new algorithm is evaluated based on protein standards with 48 known proteins. The evaluation shows better peak detection accuracy for low abundance proteins than other LC/MS peak detection methods. PMID:21143790

Molluscan mega-hemocyanin: an ancient oxygen carrier tuned by a ~550 kDa polypeptide

PubMed Central

2010-01-01

Background The allosteric respiratory protein hemocyanin occurs in gastropods as tubular di-, tri- and multimers of a 35 × 18 nm, ring-like decamer with a collar complex at one opening. The decamer comprises five subunit dimers. The subunit, a 400 kDa polypeptide, is a concatenation of eight paralogous functional units. Their exact topology within the quaternary structure has recently been solved by 3D electron microscopy, providing a molecular model of an entire didecamer (two conjoined decamers). Here we study keyhole limpet hemocyanin (KLH2) tridecamers to unravel the exact association mode of the third decamer. Moreover, we introduce and describe a more complex type of hemocyanin tridecamer discovered in fresh/brackish-water cerithioid snails (Leptoxis, Melanoides, Terebralia). Results The "typical" KLH2 tridecamer is partially hollow, whereas the cerithioid tridecamer is almost completely filled with material; it was therefore termed "mega-hemocyanin". In both types, the staggering angle between adjoining decamers is 36°. The cerithioid tridecamer comprises two typical decamers based on the canonical 400 kDa subunit, flanking a central "mega-decamer" composed of ten unique ~550 kDa subunits. The additional ~150 kDa per subunit substantially enlarge the internal collar complex. Preliminary oxygen binding measurements indicate a moderate hemocyanin oxygen affinity in Leptoxis (p50 ~9 mmHg), and a very high affinity in Melanoides (~3 mmHg) and Terebralia (~2 mmHg). Species-specific and individual variation in the proportions of the two subunit types was also observed, leading to differences in the oligomeric states found in the hemolymph. Conclusions In cerithioid hemocyanin tridecamers ("mega-hemocyanin") the collar complex of the central decamer is substantially enlarged and modified. The preliminary O2 binding curves indicate that there are species-specific functional differences in the cerithioid mega-hemocyanins which might reflect different physiological

2016 ASMS Workshop Review: Next Generation LC/MS: Critical Insights and Future Perspectives

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Hongying; Makarov, Alexander; Smith, Richard D.

The pilot workshop on BNext Generation LC/MS: Critical Insights and Future Perspectives was held on the evening of June 6, 2016 at the 64th ASMS Conference on Mass Spectrometry and Allied Topics held in San Antonio, TX. The workshop, chaired by Hongying Gao (Pfizer), consisted of stimulating talks from distinguished speakers and open discussion among the audience and invited presenters.The objectives of this workshop were to better understand the advances and limitations of current technologies; to exchange perspectives on the next generation LC/MS; and to discuss/debate the features of next generation LC/MS focusing on the following three questions: (1) Whatmore » would the next generation LC/MS look like? (2) How would it change the way we do analysis? and (3) What fundamental issues need to be resolved? A real-world case in the biopharmaceutical industry was presented by Hongying Gao on the needs by industry for LC/MS innovation and technology advancements. The primary invited speakers were Alexander Makarov (Thermo Fisher Scientific) and Richard (Dick) Smith (Pacific Northwest National Laboratory). The open discussions started with Q&A and comments for Alexander Makarov and Dick Smith, followed by insights and perspectives from members of the audience and other invited presenters who shared their thoughts addressing the above questions.« less

Stability of omega-3 LC-PUFA-rich photoautotrophic microalgal oils compared to commercially available omega-3 LC-PUFA oils.

PubMed

Ryckebosch, Eline; Bruneel, Charlotte; Termote-Verhalle, Romina; Lemahieu, Charlotte; Muylaert, Koenraad; Van Durme, Jim; Goiris, Koen; Foubert, Imogen

2013-10-23

Microalgae are the primary producers of omega-3 LC-PUFA, which are known for their health benefits. Their oil may thus be a potential alternative for fish oil. However, oxidative and hydrolytic stability of omega-3 LC-PUFA oils are important parameters. The purpose of this work was therefore to evaluate these parameters in oils from photoautotrophic microalgae (Isochrysis, Phaeodactylum, Nannochloropsis gaditana, and Nannochloropsis sp.) obtained with hexane/isopropanol (HI) and hexane (H) and compare them with commercial omega-3 LC-PUFA oils. When the results of both the primary and secondary oxidation parameters were put together, it was clear that fish, tuna, and heterotrophic microalgae oil are the least oxidatively stable oils, whereas krill oil and the microalgae oils performed better. The microalgal HI oils were shown to be more oxidatively stable than the microalgal H oils. The hydrolytic stability was shown not to be a problem during the storage of any of the oils.

LC-MS/MS imaging with thermal film-based laser microdissection.

PubMed

Oya, Michiko; Suzuki, Hiromi; Anas, Andrea Roxanne J; Oishi, Koichi; Ono, Kenji; Yamaguchi, Shun; Eguchi, Megumi; Sawada, Makoto

2018-01-01

Mass spectrometry (MS) imaging is a useful tool for direct and simultaneous visualization of specific molecules. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to evaluate the abundance of molecules in tissues using sample homogenates. To date, however, LC-MS/MS has not been utilized as an imaging tool because spatial information is lost during sample preparation. Here we report a new approach for LC-MS/MS imaging using a thermal film-based laser microdissection (LMD) technique. To isolate tissue spots, our LMD system uses a 808-nm near infrared laser, the diameter of which can be freely changed from 2.7 to 500 μm; for imaging purposes in this study, the diameter was fixed at 40 μm, allowing acquisition of LC-MS/MS images at a 40-μm resolution. The isolated spots are arranged on a thermal film at 4.5-mm intervals, corresponding to the well spacing on a 384-well plate. Each tissue spot is handled on the film in such a manner as to maintain its spatial information, allowing it to be extracted separately in its individual well. Using analytical LC-MS/MS in combination with the spatial information of each sample, we can reconstruct LC-MS/MS images. With this imaging technique, we successfully obtained the distributions of pilocarpine, glutamate, γ-aminobutyric acid, acetylcholine, and choline in a cross-section of mouse hippocampus. The protocol we established in this study is applicable to revealing the neurochemistry of pilocarpine model of epilepsy. Our system has a wide range of uses in fields such as biology, pharmacology, pathology, and neuroscience. Graphical abstract Schematic Indication of LMD-LC-MS/MS imaging.

2A4 binds soluble and insoluble light chain aggregates from AL amyloidosis patients and promotes clearance of amyloid deposits by phagocytosis †.

PubMed

Renz, Mark; Torres, Ronald; Dolan, Philip J; Tam, Stephen J; Tapia, Jose R; Li, Lauri; Salmans, Joshua R; Barbour, Robin M; Shughrue, Paul J; Nijjar, Tarlochan; Schenk, Dale; Kinney, Gene G; Zago, Wagner

2016-09-01

Amyloid light chain (AL) amyloidosis is characterized by misfolded light chain (LC) (amyloid) deposition in various peripheral organs, leading to progressive dysfunction and death. There are no regulatory agency-approved treatments for AL amyloidosis, and none of the available standard of care approaches directly targets the LC protein that constitutes the amyloid. NEOD001, currently in late-stage clinical trials, is a conformation-specific, anti-LC antibody designed to specifically target misfolded LC aggregates and promote phagocytic clearance of AL amyloid deposits. The present study demonstrated that the monoclonal antibody 2A4, the murine form of NEOD001, binds to patient-derived soluble and insoluble LC aggregates and induces phagocytic clearance of AL amyloid in vitro. 2A4 specifically labeled all 21 fresh-frozen organ samples studied, which were derived from 10 patients representing both κ and λ LC amyloidosis subtypes. 2A4 immunoreactivity largely overlapped with thioflavin T-positive labeling, and 2A4 bound both soluble and insoluble LC aggregates extracted from patient tissue. Finally, 2A4 induced macrophage engagement and phagocytic clearance of AL amyloid deposits in vitro. These findings provide further evidence that 2A4/NEOD001 can effectively clear and remove human AL-amyloid from tissue and further support the rationale for the evaluation of NEOD001 in patients with AL amyloidosis.

2A4 binds soluble and insoluble light chain aggregates from AL amyloidosis patients and promotes clearance of amyloid deposits by phagocytosis †

PubMed Central

Renz, Mark; Torres, Ronald; Dolan, Philip J.; Tam, Stephen J.; Tapia, Jose R.; Li, Lauri; Salmans, Joshua R.; Barbour, Robin M.; Shughrue, Paul J.; Nijjar, Tarlochan; Schenk, Dale; Kinney, Gene G.; Zago, Wagner

2016-01-01

Abstract Amyloid light chain (AL) amyloidosis is characterized by misfolded light chain (LC) (amyloid) deposition in various peripheral organs, leading to progressive dysfunction and death. There are no regulatory agency–approved treatments for AL amyloidosis, and none of the available standard of care approaches directly targets the LC protein that constitutes the amyloid. NEOD001, currently in late-stage clinical trials, is a conformation-specific, anti-LC antibody designed to specifically target misfolded LC aggregates and promote phagocytic clearance of AL amyloid deposits. The present study demonstrated that the monoclonal antibody 2A4, the murine form of NEOD001, binds to patient-derived soluble and insoluble LC aggregates and induces phagocytic clearance of AL amyloid in vitro. 2A4 specifically labeled all 21 fresh-frozen organ samples studied, which were derived from 10 patients representing both κ and λ LC amyloidosis subtypes. 2A4 immunoreactivity largely overlapped with thioflavin T–positive labeling, and 2A4 bound both soluble and insoluble LC aggregates extracted from patient tissue. Finally, 2A4 induced macrophage engagement and phagocytic clearance of AL amyloid deposits in vitro. These findings provide further evidence that 2A4/NEOD001 can effectively clear and remove human AL-amyloid from tissue and further support the rationale for the evaluation of NEOD001 in patients with AL amyloidosis. PMID:27494229

Genetic Diversity and Natural Selection in 42 kDa Region of Plasmodium vivax Merozoite Surface Protein-1 from China-Myanmar Endemic Border.

PubMed

Zhou, Xia; Tambo, Ernest; Su, Jing; Fang, Qiang; Ruan, Wei; Chen, Jun-Hu; Yin, Ming-Bo; Zhou, Xiao-Nong

2017-10-01

Plasmodium vivax merozoite surface protein-1 (PvMSP1) gene codes for a major malaria vaccine candidate antigen. However, its polymorphic nature represents an obstacle to the design of a protective vaccine. In this study, we analyzed the genetic polymorphism and natural selection of the C-terminal 42 kDa fragment within PvMSP1 gene (Pv MSP142) from 77 P. vivax isolates, collected from imported cases of China-Myanmar border (CMB) areas in Yunnan province and the inland cases from Anhui, Yunnan, and Zhejiang province in China during 2009-2012. Totally, 41 haplotypes were identified and 30 of them were new haplotypes. The differences between the rates of non-synonymous and synonymous mutations suggest that PvMSP142 has evolved under natural selection, and a high selective pressure preferentially acted on regions identified of PvMSP133. Our results also demonstrated that PvMSP142 of P. vivax isolates collected on China-Myanmar border areas display higher genetic polymorphisms than those collected from inland of China. Such results have significant implications for understanding the dynamic of the P. vivax population and may be useful information towards China malaria elimination campaign strategies.

Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF.

PubMed

Jiang, Hui; Sidhu, Rohini; Fujiwara, Hideji; De Meulder, Marc; de Vries, Ronald; Gong, Yong; Kao, Mark; Porter, Forbes D; Yanjanin, Nicole M; Carillo-Carasco, Nuria; Xu, Xin; Ottinger, Elizabeth; Woolery, Myra; Ory, Daniel S; Jiang, Xuntian

2014-07-01

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and "dilute and shoot" procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

A computer method of finding valuations forcing validity of LC formulae

NASA Astrophysics Data System (ADS)

Godlewski, Łukasz; Świetorzecka, Kordula; Mulawka, Jan

2014-11-01

The purpose of this paper is to present the computer implementation of a system known as LC temporal logic [1]. Firstly, to become familiar with some theoretical issues, a short introduction to this logic is discussed. The algorithms allowing a deep analysis of the formulae of LC logic are considered. In particular we discuss how to determine if a formula is a tautology, contrtautology or it is satisfable. Next, we show how to find all valuations to satisfy the formula. Finally, we consider finding histories generated by the formula and transforming these histories into the state machine. Moreover, a description of the experiments that verify the implementation are briefly presented.

17. INTERIOR OF NORTH BEDROOM SHOWING 6LIGHT OVER 1LIGHT, DOUBLEHUNG, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

17. INTERIOR OF NORTH BEDROOM SHOWING 6-LIGHT OVER 1-LIGHT, DOUBLE-HUNG, WOOD-FRAME WINDOWS IN PAIRED ARRANGEMENT ON NORTH WALL AND SINGLY ON EAST WALL. VIEW TO NORTHEAST. - Bishop Creek Hydroelectric System, Plant 4, Worker Cottage, Bishop Creek, Bishop, Inyo County, CA

33 CFR 67.05-1 - Arrangement of obstruction lights.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Arrangement of obstruction lights... for Lights § 67.05-1 Arrangement of obstruction lights. (a) Structures having a maximum horizontal... light visible for 360°. (b) Structures having a maximum horizontal dimension of over 30 feet, but not in...

33 CFR 67.05-1 - Arrangement of obstruction lights.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Arrangement of obstruction lights... for Lights § 67.05-1 Arrangement of obstruction lights. (a) Structures having a maximum horizontal... light visible for 360°. (b) Structures having a maximum horizontal dimension of over 30 feet, but not in...

33 CFR 67.05-1 - Arrangement of obstruction lights.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Arrangement of obstruction lights... for Lights § 67.05-1 Arrangement of obstruction lights. (a) Structures having a maximum horizontal... light visible for 360°. (b) Structures having a maximum horizontal dimension of over 30 feet, but not in...

33 CFR 67.05-1 - Arrangement of obstruction lights.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Arrangement of obstruction lights... for Lights § 67.05-1 Arrangement of obstruction lights. (a) Structures having a maximum horizontal... light visible for 360°. (b) Structures having a maximum horizontal dimension of over 30 feet, but not in...

33 CFR 67.05-1 - Arrangement of obstruction lights.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Arrangement of obstruction lights... for Lights § 67.05-1 Arrangement of obstruction lights. (a) Structures having a maximum horizontal... light visible for 360°. (b) Structures having a maximum horizontal dimension of over 30 feet, but not in...

Light-induced Changes in Allophycocyanin 1

PubMed Central

Ohad, Itzhak; Schneider, Hans-Jörg A. W.; Gendel, Steven; Bogorad, Lawrence

1980-01-01

Several lines of evidence indicate that allophycocyanin is the previously unidentified “phycochrome” observed in extracts of blue-green algae. Fractions containing phycoerythrin, phycocyanin, and allophycocyanin and exhibiting light-induced absorbance changes were prepared from extracts of Nostoc muscorum and Fremyella diplosiphon by isoelectric focusing. Illumination of such fractions with red light (650 nanometers) causes a reduction in absorbance at 620 nm (≃1 to 2%) and an increase at 560 nm. The effect, (previously observed by Björn and Björn [1976 Physiol Plant 36: 297-304]) is reversible, upon illumination with green light (550 nm). Selective immunoprecipitation of the phycobiliproteins indicates that allophycocyanin is the photoresponsive pigment. At pH 4.0 to 4.2, allophycocyanin purified from the same algae or from Phormidium luridum exhibits a light-induced absorbance change at 620 nm, which coincides with its absorption maximum at this pH; the fluorescence emission of allophycocyanin under these conditions is at 647 nm and its S20,w is 2.28, compatible with an α1β1 polypeptide composition. At neutral pH (5.8 to 7.0), allophycocyanin aggregates have a sedimentation coefficient of 4.8 (≃α3β3) and an additional absorption peak at 640 nm appears while that at 620 nm remains unaffected. The fluorescence emission maximum of the larger aggregate is at 667 nm and the light-induced change in its absorption is shifted to 650 nm. The effect of pH changes in the range 4.0 to 7.0 on the spectral and aggregation properties of allophycocyanin is completely reversible. Changes in pH which affect allophycocyanin aggregation have parallel effects on absorption and fluorescence maxima as well as on the light-induced absorbance changes of the biliprotein. No evidence is provided to resolve whether this phycochrome plays the role of an adaptochrome. PMID:16661143

Electrically switchable photonic liquid crystal devices for routing of a polarized light wave

NASA Astrophysics Data System (ADS)

Rushnova, Irina I.; Melnikova, Elena A.; Tolstik, Alexei L.; Muravsky, Alexander A.

2018-04-01

The new mode of LC alignment based on photoalignment AtA-2 azo dye where the refractive interface between orthogonal orientations of the LC director exists without voltage and disappeared or changed with critical voltage has been proposed. The technology to fabricate electrically controlled liquid crystal elements for spatial separation and switching of linearly polarized light beams on the basis of the total internal reflection effect has been significantly improved. Its distinctive feature is the application of a composite alignment material comprising two sublayers of Nylon-6 and AtA-2 photoalignment azo dye offering patterned liquid crystal director orientation with high alignment quality value q = 0 . 998. The fabricated electrically controlled spatially structured liquid crystal devices enable implementation of propagation directions separation for orthogonally polarized light beams and their switching with minimal crosstalk.

Agro-pastoral expansion and land use/land cover (LU/LC) change dynamics in Central-western Brazil

NASA Astrophysics Data System (ADS)

Sanga-Ngoie, K.; Yoshikawa, S.; Kanae, S.

2011-12-01

In Brazil, large-scale land cover changes following extensive deforestations are expected to generate big impacts onto the climate and the environment over this area, with eventually many negative feedbacks on the global scale. Mato Grosso State, located in the central western Brazil, is known to be the Brazilian state with the highest deforestation rate. Land use/land cover (LU/LC) changes have been reported to occur over large areas in this state due to the introduction of large-scale mechanized agriculture, extensive cattle ranching and uncontrolled slash-and-burn cultivation since the 1980s. In this study, we specifically aim at doing more detailed analysis for the causes of deforestation and savannization in this area, with special attention to agriculture and cattle ranching industry at the municipal district level in this state. Using GIS techniques and remotely-sensed NOAA/AVHRR data, we created 5-year Digital Vegetation Model Maps characterizing LU/LC features for every five years during the 1981-2001 periods using the PCA first components of the NOAA/AVHRR multi-spectral data. Our results make it clear that: (1) LU/LC changes among the phases are of the following 3 major types: degradation, recovery or transition; (2) The changes in LU/LC features are concomitant with the advance of cattle ranching and corn production activities toward the northern parts of the state, and with the expansion of soybean production in the central and western Mato Grosso; (3) Most of the agro-pastoral business are found in the southern Mato Grosso where about 46% of the state's deforestation during the 1981-2001 period occurred; (4) Rates of vegetation change are larger over non-inhabited areas (56%), especially in the north, than over the populated zones in the south (42%). Moreover, this work sheds some new light on the patterns of the changes in LU/LC features (deforestation and savannization) for each municipal district of Mato Grosso. In general, the following activities

Green grasses as light harvesters in dye sensitized solar cells

NASA Astrophysics Data System (ADS)

Shanmugam, Vinoth; Manoharan, Subbaiah; Sharafali, A.; Anandan, Sambandam; Murugan, Ramaswamy

2015-01-01

Chlorophylls, the major pigments presented in plants are responsible for the process of photosynthesis. The working principle of dye sensitized solar cell (DSSC) is analogous to natural photosynthesis in light-harvesting and charge separation. In a similar way, natural dyes extracted from three types of grasses viz. Hierochloe Odorata (HO), Torulinium Odoratum (TO) and Dactyloctenium Aegyptium (DA) were used as light harvesters in dye sensitized solar cells (DSSCs). The UV-Vis absorption spectroscopy, Fourier transform infrared (FT-IR), and liquid chromatography-mass spectrometry (LC-MS) were used to characterize the dyes. The electron transport mechanism and internal resistance of the DSSCs were investigated by the electrochemical impedance spectroscopy (EIS). The performance of the cells fabricated with the grass extract shows comparable efficiencies with the reported natural dyes. Among the three types of grasses, the DSSC fabricated with the dye extracted from Hierochloe Odorata (HO) exhibited the maximum efficiency. LC-MS investigations indicated that the dominant pigment present in HO dye was pheophytin a (Pheo a).

Detection of N-(1-deoxy-d-fructos-1-yl) Fumonisins B2 and B3 in Corn by High-Resolution LC-Orbitrap MS